hemostasis and thrombosis protocols

Importantly, endothelial cells alsoproduce the natural inhibitor of tissue factor mediated coagulation, TFPI.. Factor VII is involved in the initiation of blood coagulation, forming a pl

From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols

Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ

Hemostasis has evolved to accommodate the conflicting needs of ing vascular integrity and free flow of blood in the vascular tree Given thehigh pressures that exists in arterial circulation, it is clearly important thatprocoagulant mechanisms exist that can minimize blood loss from a site ofvascular damage as rapidly as possible However, this powerful procoagulantresponse must be localized to prevent unwanted thrombosis and controlled toprevent thrombosis in the slower low-pressure venous circulation As a result

maintain-of these competing needs, hemostasis has evolved as a patchwork maintain-of related activating and inhibiting pathways that can either promote or suppressother elements of the overall process Hemostasis has therefore evolved tointegrate five major components: vascular endothelium, platelets, coagulantproteins, anticoagulant proteins, and fibrinolytic proteins The coordinatedhemostatic response ultimately produces a platelet plug, fibrin-based clot,deposition of white cells at the point of injury and activation of inflammatory,and repair processes, maintenance of blood flow, and vascular integrity

inter-2 Overview of Hemostasis

All components of the hemostatic mechanism exist under resting conditions

in an inactive form A diagrammatic representation of the overall hemostatic

response is shown in Fig 1 Following injury, there is immediate

vasoconstric-tion and reflex constricvasoconstric-tion of adjacent small arteries This slows blood flowinto the damaged area The reduced blood flow allows contact activation ofplatelets On activation by tissue injury (or other agonists), platelets undergo aseries of physical, biochemical, and morphological changes Platelets adhere

to exposed connective tissue, mediated in part by the von Willebrand factor

(vWF) Collagen exposure and local thrombin generation (see Subheading 6.)

lead to the release of platelet granule contents Release of platelet granule

con-tents, which include adenosine diphosphate (ADP), serotonin, and fibrinogen,

further enhances platelet activation, formation of platelet aggregates, and action with other platelets and leukocytes This process leads to the formation

inter-of the initial platelet plug

The vascular endothelium also undergoes a series of changes moving fromits resting phase (with predominantly anticoagulant properties) to a more activeprocoagulant and repair phase In concert with these cellular changes, inactiveplasma coagulation factors are converted to their respective active species bycleavage of one or two internal peptide bonds In sequence, these active factorsgenerate thrombin, which leads to formation of fibrin from fibrinogen (to sta-bilize the platelet plug), crosslinking of the fibrin formed (via activation offactor XIII), further activation of platelets, and also activation of fibrinolyticpathways (to enable plasmin to dissolve fibrin strands in the course of woundhealing) Additionally, thrombin interacts with other nonhemostatic systems topromote cellular chemotaxis, fibroblast growth, and wound repair

3 Components of the Hemostatic System

3.1 Vascular Endothelium

Vascular endothelium is the monolayer of cells that line the inner surface ofblood vessels Since an uninterrupted vascular tree is necessary for survival,the ability of the vasculature to maintain a nonleaking system is essential If avessel is disrupted and leakage occurs, the coagulation system and plateletsclose the defect temporarily until cellular repair of the defect takes place If avessel is occluded by thrombus, blood flow may be re-established by lysing theclot or recanalizing the occluded vessel These properties are the main func-tional characteristics of the vascular endothelial cell

Endothelial cells are attached to and rest on the subendothelium, anextracelluar matrix secreted by the endothelial cells Subendothelium is com-posed of collagen, elastin, mucopolysaccharides (including heparan sulfate,dermatan sulfate, chrondroitin sulfate), laminin, fibronectin, vWF, vitronectin,thrombospondin, and occasionally fibrin All these components are synthesized

by the endothelial cells Together, endothelium and subendothelium form a

selectively impermeable layer, resistant to the passive transfer of fluid and lular elements of blood, but permeable to gases Cells may pass through theendothelium at sites of inflammation by a process of adherence and thenmigration between endothelial cells Subendothelium can act as a physical bar-rier in the absence of endothelial cells Endothelial cells have multiple func-

cel-tions as outlined below (1).

3.1.1 Maintenance of Blood Flow

Endothelial cells influence vascular tone, blood pressure, and blood flow byinduction of vasoconstriction and vasodilatation This is achieved by secretion

of renin, endothelin, endothelial-derived relaxing factor (EDRF) or nitrousoxide, adenosine, prostacyclin, and surface enzymes that convert or inactivateother vasoactive peptides, such as angiotensin and bradykinin

3.1.2 Antiplatelet and Anticoagulant Properties

Intact endothelial cells are intrinsically nonthrombogenic, exerting a ful inhibitory influence on hemostasis by a range of factors that they eitherFig 1 A flow diagram representing the major events in the process of overallhemostasis

power-synthesize or express on their surface For example, platelets adhere tosubendothelium rather than endothelial cells This is due to endothelial pro-duction of components that inhibit platelet aggregation, such as prostacylin,EDRF, and adenosine.

Cell-surface heparan sulfate enhances the effect of antithrombin in formingthrombin–antithrombin complexes Perhaps the major anticoagulant proper-ties of endothelium are via the endothelial expression of thrombomodulin andtissue factor pathway inhibitor (TFPI) Thrombomodulin enhances the ability

of thrombin to activate protein C Enhancement of protein C activation leads toincreased inactivation of factor Va and factor VIIIa Endothelium also secretesprotease nexin 1 This inactivates thrombin by covalent binding to the throm-bin active site This complex formation is enhanced by heparan sulfate.3.1.3 Coagulant Properties

In contrast to the above, in the setting of damage to blood vessels, the endotheliumfunctions as an important component to coagulation pathways Central to this role isendothelial cells production of tissue factor in response to injury In addition, theybind factors IX, X, V, high-mol-wt kininogen (HMWK), contain factor XIII activity,and produce endothelin to induce vasoconstriction Importantly, endothelial cells alsoproduce the natural inhibitor of tissue factor mediated coagulation, TFPI

3.1.4 Fibrinolytic Properties

Endothelial cells secrete several components active in fibrinolysis These includeplasminogen activators and plasminogen activator inhibitor These components arebound to the endothelial cell surface to enable assembly of active complexes.3.1.5 Repair Properties

Endothelial cells are capable of significant repair of blood vessels Simpleminor injuries are repaired by migration of adjacent cells and subsequentendothelial cell proliferation More severe vessel wall injuries require migra-tion and proliferation of smooth muscle cells and fibroblasts Endotheliumsecretes components that are active in the repair process by enhancing smoothmuscle migration and fibroblast function These include a protein resemblingplatelet-derived growth factor, vascular permeability factor, and fibroblastgrowth factor Endothelial cells are also responsive to platelet-derived endot-helial growth factor and transforming growth factor β

3.1.6 Interactive Properties

The endothelium interacts with leukocytes This is critical in the migration

of leukocytes into area of inflammation Adhesion molecules present on bothendothelial cells and leukocytes mediate this interaction

4 Platelets

Platelets are nonnucleated fragments of cytoplasm that have a crucial role inprimary hemostasis They are derived from bone marrow megakaryocytes andare smooth biconvex disks of approx 1–4 mm diameter Normal circulatingnumbers are approx 140–400 × 109/L

4.1 Production

In the production of platelets, megakaryocytes undergo specialized cellulardivision The megakaryocyte nucleus divides, but the cell itself does not divide

(endomitosis) (2), although there is formation of new membrane and

cytoplas-mic maturation This cytoplascytoplas-mic maturation includes development of let-specific granules, membrane glycoproteins, and lysosomes Mature

plate-megakaryocytes are therefore variably polyploid, with up to 64 N They are

large at approx 60 µm diameter As a part of the endomitosis process, there isincreased membrane This excess membrane is accommodated by invagina-tion The invagination process continues, thereby clipping off individual plate-lets (cytoplasmic fragmentation) from the main megakaryocyte body It issuggested that circulating megakaryocytes undergo cytoplasmic fragmentation

in the pulmonary capillary bed

Megakaryocyte maturation is controlled in a simple negative feedback loop,under the influence of the growth factor thrombopoietin and cytokines, such asinterleukin-3 (IL-3) and interleukin-11 (IL-11) When platelet production isincreased, megakaryocytes undergo a more rapid cytoplasmic maturation thannuclear maturation Under such circumstances, platelets may be produced fromoctaploid or even tetraploid cell megakaryocytes Such platelets are often largerthan normal and more metabolically active

Once released from the bone marrow, platelets are sequestered in the spleenfor 24–48 h The spleen may contain upto 30% of the normal circulating mass

of platelets Significant platelet pools may also exist in the lungs

The normal life-span of platelets is approx 8–14 d Platelets are removedfrom the circulation by the reticuloendothelial system on the basis of senes-cence rather than by random utilization However, there is a small fixed com-ponent that exists owing to random utilization of platelets that maintainvascular integrity

4.2 Structure

Stylized structural features are shown diagrammatically in Fig 2 A range of

glycoproteins molecules partially or completely penetrate cell-membrane lipidbilayer These glycoprotein molecules function as receptors for different ago-nists, adhesive proteins, coagulation factors, and for other platelets Important

membrane glycoproteins are listed in Table 1 with their associated functions.

The most abundant glycoproteins on the platelet surface are glycoproteinsIIb and IIIa These two glycoproteins form a heterodimer and carry receptorsfor adhesive proteins (fibrinogen, vWF, fibronectin) The IIb-IIIa complex is amember of the integrin family of adhesion receptors Glycoprotein Ib contains

a receptor for vWF and thrombin This receptor is essential in the platelet sel wall interaction The cell membrane also has importance as a source ofphospholipid (prostaglandin synthesis), site of calcium mobilization, andlocalization of coagulant activity to the platelet surface

ves-Fig 2 Stylized structural features of the platelet See text for decription of vidual components

indi-Table 1

Important Platelet Membrane Glycoproteins

Platelet structure is complex (3) Below the plasma membrane lies a

periph-eral band of microtubules, which function as the cellular cytoskeleton Themicrotubules maintain the discoid shape in the resting platelet, but disappeartemporally (disassemble?) on platelet aggregation

The surface-connected canalicular system is an extensive system of plasmamembrane invaginations This system vastly increases the surface area acrosswhich membrane transport occurs and through which platelet granules dis-charge their contents during the secretory phase of platelet aggregation.The dense tubular system probably represents the smooth endoplasmicreticulum It is thought to be the site of prostaglandin synthesis and sequestra-tion/release of calcium ions

Platelets contain many organelles (mitochondria, glycogen granules, omes, peroximsomes) and two types of platelet-specific storage granules: densebodies (d-granules) or a-granules The contents of the platelet-specific gran-ules are released when platelets aggregate

lysos-Dense bodies contain 60% of the platelet storage pool of adenine otides (such as adenosine diphosphate) and serotonin Dense body adeninenucleotides do not readily exchange with other adenine nucleotides in the plate-let metabolic pool α-Granules contain multiple different proteins These pro-teins may be platelet specific or proteins that are found in the plasma or othercell types (such as coagulation factors) The major contents of α-granules arevWF, platelet factor 4, β-thromboglobulin, thrombospondin, factor V, fibrino-gen, fibronectin, platelet derived growth factor, high-mol-wt kininogen, andtissue plasminogen activator inhibitor-1

nucle-4.3 Function

Platelets are crucial components of the hemostatic system When a vesselwall is damaged, platelets escaping from the circulation immediately come intocontact with and adhere to collagen and subendothelial bound vWF (throughglycoprotein Ib) Glycoprotein IIb-IIIa is then exposed, via the binding of vWF.This forms a second binding site for vWF In addition with glycoprotein IIb–IIIa exposure, fibrinogen may be bound promoting platelet aggregation Withinseconds of adhesion to the vessel wall, platelets undergo a shape change, ow-ing to ADP released from the damaged cells or other platelets exposed to thesubendothelium Platelets become more spherical and put out pseudopods,which enable platelet–platelet interaction The peripheral microtubules becomecentrally apposed forcing the granules toward the surface and the surface-con-nected canalicular system Platelets then undergo a specific release reaction oftheir granules, the intensity of the release reaction being dependent on the in-tensity of the stimulus With the shape change, there is also further exposure ofthe glycoprotein IIb–IIIa complex and further fibrinogen binding Since fi-

brinogen is a dimer, it can form a direct bridge between platelets or act as asubstrate for the lectin-like protein thrombospondin With the enhancement ofplatelet–platelet interaction, platelet aggregation ensues Platelet aggregationcauses activation, secretion, and release from other platelets, so leading to aself-sustaining cycle that results in the formation of a platelet plug.

The binding of agonists to also leads to a series of signal transduction events

that mediate the platelet release reaction (see Fig 3) (4) Agonist receptor

interaction activates guanine nucleotide binding proteins (G-protein) andhydrolysis of plasma membrane phospholipids (phosphotidyl inositides) byphospholipase C (PLC) Inositol triphosphates that are formed act as iono-phores, and mobilize calcium ions into the cytosol from the dense tubular sys-tem, and lead to an influx of calcium from outside Diacylglycerol, also formedwithin the G-protein/PLC pathway, activates protein kinase C, which in turnphosphorylates a 47-kDa contractile protein Together with the calcium-dependent phosphorylation of myosin light chain, these reactions induce con-traction and secretion of granule contents Cyclic AMP/adenyl cyclase exertregulatory control over these reactions (high levels of cAMP reduce cytosolcalcium concentration) and are in turn regulated by G-protein activity In addi-tion, prostaglandin (cyclic endoperoxides and thromboxane A2) synthesizedfrom membrane phospholipids may bind to specific receptors and furtherstimulate these processes

Platelet α-granules contain several coagulation factors (such as factor V,fibrinogen, and high-mol-wt kininogen) On secretion from the α-granule, thesefactors reach high local concentrations Platelets provide a local phospholipidsurface for these factors to work on, particularly factor V This procoagulantactivity of platelets is not seen in resting platelets

4.4 Antigens

Platelets have a number of antigens on their surface specific to platelets.Many of the platelet-specific antigens are associated with platelet membraneglycoproteins (HPA IA—glycoprotein IIIa) Platelets also express HLA class Iantigens and ABO blood group antigens

5 Coagulation Factors

5.1 Thrombin

Thrombin is the cornerstone of hemostasis Prothrombin, its precursor, is avitamin K dependent plasma of mol wt 71 kDa (579 amino acids) Thrombin iscrucial to the conversion of fibrinogen to fibrin It is the most potent physi-ological activator of platelets causing shape change, the generation of throm-boxane A2, ADP release, and ultimately platelet aggregation Thrombin alsoactivates the cofactors of coagulation factor V, factor VIII, and factor XIII

Thrombin bound to thrombomodulin is a potent activator of protein C In addition

to its procoagulant and anticoagulant activities, thrombin also has important roles

in cellular growth, cellular activation, and the regulation of cellular migration

5.2 Tissue Factor

This is an integral transmembrane protein of mol wt 45 kDa (263 aminoacids) coded for by a short gene of 12.4 kb on chromosome 1 It is found on thesurface of vascular cells, but is also constitutively expressed by manynonvascular tissues It can be upregulated on monocytes and vascular endothe-lium by inflammatory cytokines or endotoxin Tissue factor (thromboplastin)binds and promotes activation of factor VII, and is required for the initiation ofblood coagulation It acts as a cofactor enhancing the proteolytic activity offactor VIIa toward factor IX and factor X It binds factor VII via calcium ions

in coagulation, which in its activated form facilities the conversion of thrombin to thrombin In Factor V, the rate of conversion of prothrombin tothrombin is 200,000- to 300,000-fold.

pro-Factor V circulates as a single-chain protein in a precursor inactive form It isconverted into an active two-chain form by thrombin or factor Xa Thrombin cleavesfactor V at three separate sites Following cleavage, the two chains are linked via adivalent metal ion bridge Binding to phospholipid surfaces occurs via the light chain.Factor V is inactivated by activated protein C and its cofactor protein S

Although it is predominantly synthesized in the liver (plasma factor V),megakaryocytes also synthesize factor V, which is stored in platelet α-gran-ules (platelet factor V) Platelet factor V, which is released on platelet activa-tion, accounts for approx 20% of total factor V Factor V has a binding protein

in platelets (multimerin), which is analogous to vWF for factor VIII

Plasma concentration of factor V is about 7–10 µg/mL with a half-life ofapprox 12 h

5.4 Factor VII

This is a vitamin K dependent plasma glycoprotein and serine protease ofmol wt 50 kDa (406 amino acids) coded for by a 13-kb gene on chromosome

13 It has 10 N-terminal glutamic acid residues that are terminal γ carboxylated

to form the Gla domain Calcium binding properties of factor VII are crucial toits normal function and biological activity

Factor VII is involved in the initiation of blood coagulation, forming a plex with tissue factor to generate an enzyme complex that activates factor Xand factor IX Factor VII is activated by cleavage of the Arg153–Ile153 peptidebond Activators include thrombin, activated factor X, and activated factor IX.Activated factor VII has no catalytic activity until bound to tissue factor Itcirculates at a concentration of 0.5 µg/mL and half-life of 4–6 h

com-5.5 Factor VIII

This is a plasma glycoprotein of approx mol wt 360 kDa (2351 amino acids)coded for by a complex 26 exon 186-kb gene on the X chromosome It has adomain structure that is very similar to that of factor V and is related to thecopper protein ceruloplamsin However, unlike factor V, the large B domain isnot required for coagulant activity Factor VIII is one of the largest and leaststable coagulation factors with a complex polypeptide composition, circulat-ing in plasma in a noncovalent complex with vWF vWF functions to protectfactor VIII from premature proteolytic degradation and concentrate factor VIII

at sites of vascular injury Factor VIII functions as a cofactor for factor IX,facilitating the conversion of factor X to factor Xa Factor VIII increases therate of conversion of factor X to Xa by factor IX by 200,000-fold

Liver synthesized single-chain molecule factor VIII is cleaved shortly aftersynthesis, circulates as a heterodimer, and comprises an 80-kDa light chainlinked through a divalent metal cation bridge to a heavy chain (90–200 kDa).Variable amounts of the B domain remain after this initial cleavage On activa-tion by thrombin (or factor Xa), factor VIII is cleaved at Arg372, Arg740, andArg1689, the Arg740cleavage removing residual B domain remnants This cleav-age yields a 90-kDa heavy chain A rate-limiting Arg372cleavage yields twosmaller 50 and 40 kDa fragments, both of which are essential for factor VIIIclotting activity At the same time, a small fragment is cleaved that removesvWF from factor VIII Activated factor VIII is very unstable and rapidly losescofactor function, owing to subunit disassociation Inactivation of factor VIIIalso occurs via activated protein C and its cofactor protein S, by cleavage atArg336and Arg562 Plasma concentration of factor VIII is about 100–200 ng/mLand half-life of approx 12 h.

5.6 Factor X

This is a vitamin K-dependent plasma glycoprotein and serine protease ofmol wt 59,000 coded for by a 22-kb gene on chromosome 13 It has 11N-terminal glutamic acid residues that are terminal γ carboxylated to form theGla domain Calcium binding properties of factor X are crucial to its normalfunction and biological activity It is a central component in the common path-way of blood coagulation Factor X is synthesized as a single chain, but exists

in plasma as a heavy and light chain linked by a single disulfide bond It isactivated by cleavage of the Arg51–Ile52 peptide bond Activators includeactivated factor VII/tissue factor complex and activated factor IX/factor VIIIcomplex in the presence of calcium ions

Factor Xa, in conjunction forms a complex on phospholipid surfaces withfactor V to form the prothrombinase complex This complex converts pro-thrombin to thrombin Factor X is inhibited by antithrombin and α2macro-globulin

Factor X circulates at a concentration of 8–10 µg/mL and has a half-life ofapprox 36 h

Factor IX circulates as a single-chain polypeptide Activation occurs via age of two peptide bonds, Arg145–Ala146and Arg180–Val181, by either activatedfactor XI or activated factor VII, complexed to tissue factor Arg180–Val181cleav-age is rate-limiting Cleavage into factor IXa generates a heavy and light chainbound together via a single disulfide bond A 24 amino acid activation peptide isremoved during cleavage Together with factor VIII, factor IXa can then proceed

cleav-to activate faccleav-tor X In addition, faccleav-tor IXa may also activate faccleav-tor VII.The plasma factor IX concentration is about 5 µg/mL with a half-life ofapprox 24 h

5.8 Factor XI

This is plasma glycoprotein and serine protease of mol wt 160 kDa coded by

a 23-kb gene on chromosome 4 Factor XI is a homodimer, comprising twoidentical subunits bound together by disulfide bond, that circulates bound tohigh-mol-wt kininogen The plasma factor XI concentration is about 5 µg/mLwith a half-life of approx 72 h

Factor XI is cleaved to active factor XIa by activated factor XII in the ence of high-mol-wt kininogen Activation cleavage occurs within each sub-unit at Arg369–Ile370in a region bounded by a disulfide linkage, so yielding twoheavy chains and two light chains in the active molecule Only the light chainspossess catalytic activity Factor XIa activates factor IX in the presence of cal-cium No specific additional cofactors are required for this reaction Both fac-tor XI and factor XIa bind to platelets

pres-5.9 Factor XII

This is a plasma glycoprotein and serine protease of mol wt 80 kDa (596amino acids) coded for by a 12-kb gene located on chromosome 5 Factor XIIhas a half-life of approx 2 d and a plasma concentration of approx 30 µg/mL

In the process of contact activation factor XII is absorbed on to negativelycharged surfaces and undergoes limited proteolysis at specific sites to yieldactive factor XII' This slowly converts prekallikrein to kallikrein, which spe-cifically cleaves factor XII to yield fully active factor XIIa In addition, factorXIIa can autoactivate factor XII Factor XIIa can activate factor XI to promotedownstream activation of the coagulation cascade

5.10 Factor XIII

This is a tetramer of two a and b chains The b chains function as the carrierfor the a chains On activation by thrombin, factor XIII crosslinks fibrin andother proteins involved in the clot via a transglutamase reaction The factorXIIIa subunit has a plasma concentration of 15 µg/mL and the b subunit aconcentration of 14 µg/mL

5.11 von Willebrand Factor (vWF)

This is a multimeric glycoprotein of basic subunit of 400 kDa mol wt (2813amino acids) coded for by a large complex gene of 178-kb on the short arm ofchromosome 12 vWF is an important component in primary platelet hemosta-sis Following translation, it undergoes extensive intracellular processing andexists as a series of multimers of the basic subunit, ranging from mol wt 800 to20,000 kDa It is produced in both endothelium and megakaryocytes Endothe-lial cells secrete a vWF into the plasma constitutively, but store the majority ofvWF synthesized (in Wieble Palade bodies) for regulated secretion PlateletvWF is released from α-granules locally when they aggregate vWF functions:

as a carrier protein for coagulation factor VIII and as an adhesive proteininvolved in endothelial-platelet interaction, via platelet surface membrane gly-coprotein Ib and IIb–IIIa complex Its function as an adhesive protein is par-ticularly important in situations of high shear stress

6 Coagulation Cascade

The classic “waterfall” hypothesis for coagulation proposes the intrinsic and

extrinsic pathways (see Fig 4) (5,6) The intrinsic system assumes that

expo-sure of contact factors (factor XII, high-mol-wt kininogens, prekallikrein) to

an abnormal/injured vascular surface leads to activation of factor XI, which inturn activates factor IX Activated factor IX, in the presence of its cofactorfactor VIII, then activates factor X to factor Xa in the presence of phospho-lipid In turn, factor Xa, with its cofactor factor V, together form theprothrombinase complex, which converts prothrombin to thrombin Thrombinthen converts fibrinogen to fibrin The extrinsic system assumes that factor VIIand tissue factor, released from damaged vessels, directly activate factor X,and coagulation factor lying below factor X in the final common pathway.The division into extrinsic and intrinsic systems and the ability to test thesetwo systems in the laboratory (the prothrombin time and activated partialthromboplastin time, respectively) have been valuable in understanding clini-cal bleeding problems, but fail to represent accurately what happens in in vivohemostasis This may be shown by considering the following points First,patients who have an inherited deficiency of factor XII, prekallikrein or high-mol-wt kininogen have no clinical bleeding problems, yet have extremely pro-longed activated partial thromboplastin times This clinical observationdemonstrates that these proteins are probably not important components ofblood coagulation in vivo and, therefore, should not be included in an in vivoconsideration of blood coagulation Similarly, factor XI deficiency is notalways associated with bleeding and its role is therefore unclear, whereaspatients with factor VII deficiency bleed abnormally, although they have anintact intrinsic system Third, factor VII–tissue factor is known to activate not

only factor X, but also factor IX In the classic waterfall, this activation is notrequired Fourth, tissue factor is a natural constituent of many nonvascular cells.Tissue factor on such cells is able to initiate blood coagulation These pointssuggest a more central role for the tissue factor–factor VII complex Addition-ally, the identification of an endogenous inhibitor of tissue factor-inducedcoagulation (tissue factor pathway inhibitor; TFRI) and an increased understand-

ing of its properties have led to a questioning of traditional dogmas (7).

The revised cascade is outlined in Fig 5 This revised cascade is believed to

represent more accurately the processes that occur in vivo (7–9) Coagulation

is initiated when tissue damage at the site of the wound exposes blood to tissuefactor, produced constitutively by cells beneath the endothelium Factor VIIbinds tissue factor forming the tissue factor–factor VII complex This complexdirectly activates factor X to factor Xa and some factor IX to factor IXa It isnot clear what proteases initially activate factor VII in this complex, but oncecoagulation is activated, other proteins are able in turn to activate factor VII,including factor Xa and VIIa This provides a mechanism for further amplifi-cation and acceleration of coagulation

Fig 4 Classic “waterfall” hypothesis for coagulation with the intrinsic, extrinsic,and final common pathways Although useful in understanding coagulation pathways

in in vitro clotting assays this schema does not accurately represent in vivo tion processes

coagula-Once formed, the complex of the factor VIIa, tissue factor, and factor Xabinds TFPI forming a quaternary complex This TFPI binding inhibits furthergeneration of factor Xa and factor IXa by tissue factor–factor VIIa complex.Under these conditions, further factor Xa can only be generated by the factorIXa, factor VIIIa pathway.

By this point in coagulation activation, enough thrombin usually exists to beable to activate factor VIII to factor VIIIa (generated by direct activation offactor Xa by factor VII–tissue factor) With activation of factor VIIIa and usingthe initial generation of factor IXa (by tissue factor–factor VIIa), the factorIXa, factor VIIIa route is able to move forward and allow further factor Xageneration to proceed

Further augmentation of factor IX activation is produced via thrombin vation of the factor XI pathway This is proposed to be a process that occurslater in coagulation

acti-The revised cascade assumes that tissue factor–factor VIIa is responsible forthe initial generation of factor Xa and thrombin, sufficient to activate factor V,factor VIII, and platelet aggregation locally Following inhibition by TFPI, theFig 5 The revised coagulation pathway See text for details Note the central role

of TFPI and absence of contact factors

amount of factor Xa produced is insufficient to maintain coagulation, and fore, factor Xa generation must be amplified using factor IXa and factor VIIIa

there-to allow hemostasis there-to progress there-to completion

Unlike the waterfall hypothesis, the revised hypothesis does not assume thatinitial generation of factor Xa and thrombin is the end of the hemostatic pro-cess Rather it assumes that following initial generation the hemostatic responsemust be reinforced and/or consolidated by a further progressive generation offactor Xa and thrombin This allows the hypothesis to encompass the compet-ing influences of inhibitors of coagulation, blood flow washing away activatedcoagulation factors, and also thrombin-activated fibrinolysis Additionally, itdoes not all factor known to be involved in blood coagulation

The revised hypothesis also allows a better explanation of bleeding seen inhemophilia A and hemophilia B In these two conditions, bleeding occurs bothspontaneously (intrinsic system) and after trauma (extrinsic system), whichcannot easily be reconciled on the classical waterfall hypothesis Using therevised schema, it is clear that without factor VIII or factor IX, bleeding willensue because the amplification and consolidating generation of factor Xa isinsufficient to sustain hemostasis

7 Anticoagulant Pathways

Natural, physiological anticoagulants fall into two broad categories, serineprotease inhibitors (SERPINS) and those that neutralize specific activatedcoagulation factors (protein C system) These systems are of major physiologi-cal significance They are active from the very outset of the coagulation pro-cess and often brought fully into play before fibrin deposition has occurred

7.1 Serine Protease Inhibitors (SERPINS)

Serpins include many of the key inhibitors of coagulation, such as bin, heparin cofactor II, protein C inhibitor, plasminogen inactivators, and α2-

antithrom-antiplasmin Of these, antithrombin is perhaps the most important (10) AT is a

single-chain glycoprotein of mol wt 58,000 (432 amino acids) coded for onchromosome 1 It will inhibit all the coagulation serine proteases (II, VII, IX,

X, XI, XII), but it is its antithrombin and anti-Xa activity that are cally important AT activity/inhibition is increased 5- to 10,000-fold in thepresence of heparin and other sulfated glycosaminoglycans Heparin is notnormally found in the circulation, and physiologically, antithrombin probablybinds to heparan sulfate on the vascular endothelial cells

physiologi-7.2 Protein C System

Factors Va and VIIIa are powerful cofactors in coagulation-enhancingactivity of serine proteases Both Va and VIIIa are specifically inactivated by

components of the protein C pathway Protein C is the key inactivating enzyme

(11) It is a single-chain vitamin K-dependent protein synthesized by the liver.

Together with its cofactor, protein S it inactivates factors Va and VIIIa.Thrombin generated during coagulation binds to thrombomodulin (Tm) onthe surface of vascular endothelial cells Thrombin/Tm complex is a potentactivator of Protein C, Tm accelerating Protein C activation approx 20,000-fold Protein C is activated by cleavage at Arg169–Leu170 Activated protein C(APC) is inhibited by the specific inhibitor, protein C Inhibitor (PCI) and since

it is a serine protease, it is inhibited by antithrombin

Protein S, the cofactor for protein C, is vitamin K-dependent It circulates inplasma as a single-chain glycoprotein of mol wt 60,000 and is synthesized bythe liver, endothelial cells, and megakaryocytes Approximately 60% of pro-tein S is complexed to C4b binding protein, Only the unbound or “free” protein

S is physiologically active

8 Fibrinolysis

Fibrinolysis principally exists to ensure that fibrin deposition in excess ofthat which is required to prevent blood loss from damaged vessels is either

prevented or degraded and removed (12).

Plasminogen, the inactive form of the enzyme plasmin, has a mol wt of 92kDa (790 amino acids), and is synthesized in the liver and coded on chromo-some 6 Plasminogen contains five homologous looped structures called

“kringles,” four of which contain lysine binding sites through which the ecule interacts with its substrates and its inhibitors Internal autocatalytic cleav-age occurs during activation of plasminogen with the release of an activationpeptide This changes the N-terminus from containing a glutamic acid residue(Glu-plasminogen) to a form that contains a lysine residue (Lys-plasminogen).Conversion of plasminogen to plasmin can occur via two routes Most acti-vators cleave plasminogen at Arg560to generate a two-chain protein Glu-plas-min, the two chains linked by a single disulfide bridge The light chain isderived from the C-terminus of the protein and contains the active serine cata-lytic site, whereas the heavy chain is derived from the N-terminus and containsthe kringle domains Glu-plasmin is functionally inactive, since its lysine bind-ing sites are masked It is activated when it is converted to Lys-plasmin byautocatalytic cleavage between Lys76–Lys77 This cleavage exposes the lysinebinding sites on the kringle domains, dramatically increasing the affinity of theprotease for fibrin Both Glu-plasmin and Lys-plasmin attack the Lys76-Lys77bond to form Lys-plasminogen This is capable of binding to the fibrin clotbefore it develops protease activity, and it is, therefore brought into close prox-imity with the physiological activators

mol-Plasminogen is activated by a number of endogenous proteins Of these, sue plasminogen activator (t-PA) is perhaps the most important t-PA is synthe-

tis-sized primarily by vascular endothelial cells although many other cells, arecapable of its synthesis t-PA is synthesized as a single-chain glycoprotein (sct-PA) and contains two kringle domains through which it binds fibrin Althoughsct-PA has significant proteolytic activity, its biological activity is low untilbound to fibrin When bound, its affinity for plasminogen is increased approx400-fold Plasmin generated is capable of cleaving sct-PA into a two-chain tPA.Two-chain t-PA has more exposed binding sites and a significantly increasedactivity through increased binding of fibrin and plasminogen t-PA has a shorthalf-life (5 min) and is rapidly cleared from the circulation.

sct-PA and tct-PA are inhibited by the SERPIN plasminogen activatorinhibitor type 1 (PAI-1) A second inhibitor of t-PA, plasminogen activatorinhibitor type 2 (PAI-2) is found in plasma in significant amounts during preg-nancy Normally free t-PA is rapidly inactivated because of an excess of PAI-

1 and any free plasmin generated is rapidly inactivated by α2-antiplasmin

A second endogenous activator of fibrinolysis is urokinase Urokinase issynthesized as an essentially inactive single-chain protein (scu-PA/pro-uroki-nase) It must be converted to the two-chain form (tcu-PA or U-PA) before it isfunctionally active scu-PA is converted to tcu-PA (U-PA) by plasmin and kal-likrein tcu-PA activates plasminogen to plasmin by a cleaving at Arg560–Val561 Inhibition of the active enzyme occurs via PAI-1, PAI-2, and also byprotease nexin 1 Although urokinase can activate plasminogen in plasma it isthought that its major role is an extravascular activator of plasminogen, espe-cially where tissue destruction or cell migration occurs

9 Summary

Hemostasis is clearly a complex interactive system involving numerous nents The revised hypothesis of coagulation has helped to unify the whole pro-cess The recent improved understanding has in part been brought about byimproved knowledge of the individual components of the different elements of theoverall process of hemostasis and cellular repair Although increasing by appreci-ated to be complex, attempts have been made to model and reproduce this system

compo-in vitro to validate research fcompo-indcompo-ings and compo-increase understandcompo-ing of the compo-interactions.For all its complexity, many of these models of hemostasis, both laboratory andmathematical, have proven to be useful, and show that for all the interactions andcomplexity of different systems combined with flow and cellular interaction, we

do have a considerable understanding of the processes of hemostasis

Suggested Reading

Bloom, A L., Forbes, C D., Thomas, D P., and Tuddenham, E G D (1994)

Haemostasis and Thrombosis, 3rd ed Churchill Livingstone, Edinburgh, UK.

Tuddenham, E G D and Cooper, D N The Molecular Genetics of Haemostasis and

its Inherited Disorders Oxford Medical Publications, Oxford, UK.

1 Cines, D B., Pollak, E S., Buck, C A., Loscalzo, J., Zimmerman, G A., McEver,

R P., Pober, J S., Wick, T M., Konkle, B A., Schwartz, B S., Barnathan, E S.,McCrae, E R., Hug, B A., Schmidt, A S., and Stern, D M (1998) Endothelial

cells in physiology and in the pathophysiology of vascular disorders Blood 91,

3527–3561

2 Gerwitz, A (1995) Megakaryocytopoiesis: the state of the art Thomb.

Haemostasis 74, 204–209.

3 White, J G (1994) Anatomy and structural organisation of the platelet, in

Hemo-stasis and Thrombosis: Basic Principles and Clinical Practice (Coleman, R W.,

et al., eds.), 3rd ed., J B Lippencott Co., Philadelphia, PA

4 Levy-Toledano, S., Gallet, C., Nadel, F., Bryckaert, M., Macloug, J., and Rosa,J.-P (1997) Phosphorylation and dephosporylation mechanisms in platelet func-

tion: a tightly regulated balance Thromb Haemostasis 78, 226–233.

5 Macfarlane, R G (1964) An enzyme cascade in the blood clotting mechanism

and its function as a biochemical amplifier Nature 202, 498,499.

6 Davie, E W and Ratnoff, O D (1964) Waterfall sequence for intrinsic blood

clotting Science 145, 1310–1312.

7 Broze, G J Jr., Warren, L A., Novotny, W F., Higuchi, D A., Girrad, J J., andMiletich, J P (1988) The lipoprotein associated coagulation inhibitor that inhib-its the factor VII-tissue factor complex also inhibits factor Xa: insight into its

possible mechanism of action Blood 71, 335–343.

8 Furie, B and Furie, B C (1992) Molecular and cellular biology of blood

coagualtion N Engl J Med 326, 800–806.

9 Rapaport, S I and Rao, L V (1995) The tissue factor pathway: how it has bevome

a “prima ballerina.” Thromb Haemostasis 74, 7–17.

10 Perry, D J (1994) Antithrombin and its inherited deficiencies Blood Rev 8, 35–37.

11 Dadhlback, B (1995) New molecular insights into the genetivcs of thrombophilia:resistance to activated Protein C caused by the Arg506to Gln mutation in factor V as

a pathogenic risk factor for venous thrombosis Thromb Haemostasis 74, 139–148.

12 Collen, D and Lijen, H R (1991) Basic and clinical aspects of fibrinolysis and

thrombolysis Blood 78, 3114–3124.

From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols

Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ

There are numerous methods for isolating DNA The methods described inthis Chapter are routinely used to prepare high molecular weight DNA forSouthern blot analysis or for amplification by the polymerase chain reaction(PCR) technique The first method employs a phenol/chloroform step to dena-ture proteins, whereas the second employs a salt precipitation step to precipi-tate proteins Both methods can be readily adapted to processing small-volumesamples (e.g., 100 µL) The first method has been successfully used to isolateDNA from a wide variety of cells, including whole blood, buffy coats, plate-lets, various cell lines, spleen, lymph nodes, and bone marrow

As with the isolation of DNA, there are many techniques for isolating RNAfrom a wide variety of cells, some of which can be adapted to allow the simul-taneous isolation of both RNA and DNA Many of the methods in current useemploy strong chaotropic agents (e.g., guanidinium thiocyanate) to disrupt cell-ular membranes and inactivate intracellular RNases The method described hasbeen in routine use for several years and generates high-quality RNA suitablefor a wide variety of uses A number of commercial kits are now available forrapid RNA isolation, e.g, RNeasy™ kit (Qiagen Ltd, UK) Although often moreexpensive than “in-house” methods, these kits are capable of isolating high-

quality RNA from a wide variety of cells, including whole-blood, leukocytebuffy coats, platelets, and tissue-culture cells.

Methods are also included in this chapter for the isolation of lymphocytesand platelets from whole blood

2 Materials

Molecular-grade reagents should be used whenever possible Sterile able polypropylene is used for most steps, but if glassware is used, it should bebaked at 280°C for at least 3 h to inactivate any RNases

dispos-2.1 Isolation of Mononuclear Cells from Whole Blood

1 Phosphate-buffered saline: 120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer,

pH 7.4 Autoclave before use and store at 4°C

2 Density gradient medium, e.g., Histopaque 1077 (Sigma)

2.2 Isolation of Platelets from Whole Blood

1 Phosphate-buffered saline: 120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer,

pH 7.4 Autoclave before use and store at 4°C

2 38–40% Bovine serum albumin (BSA)

2.3 DNA Isolation Using the Phenol/Chloroform Method (1)

1 Sucrose lysis buffer: sucrose 0.32 M, 1% (v/v) Triton X-100, 5 mM MgCl2, 10 mM

Tris-HCl, pH 7.5 Store at 4°C

2 TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 Autoclave before use.

3 Nuclear lysis buffer: 0.32 M lithium acetate, 2% (w/v) lithium dodecyl sulfate (LiDS), 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 Store at room temperature.

4 Phenol: Equilibrated phenol for both DNA and RNA isolation can be purchasedcommercially (e.g., CamLabs, Cambridge, UK) and avoids many of the potentialrisks associated with its use If crystalline phenol is used, 0.1% hydroxyquinoline

should be added as an antioxidant and it should be extracted initially with 1 M Tris-HCl, pH 8.0, and then repeatedly with 0.1 M Tris-HCl, pH 8.0, until the pH

of the aqueous phase is 8.0 (2) Phenol should be stored at 4°C

5 Phenol:chloroform: A 1:1 mixture phenol and chloroform is made by mixingequal volumes of chloroform and equilibrated phenol This may be purchasedready prepared from a number of manufacturers (e.g., CamLabs) Store at 4°C

2.4 DNA Isolation Using the Salt-Precipitation Method (3)

1 Sucrose lysis buffer–see Subheading 2.3.1.

2 TKM 1: low-salt buffer–10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM MgCl2,

5 20% SDS: Dissolve 20 g SDS in 100 mL distilled water at 65°C Store at roomtemperature.

2.5 Isolation of Total Cellular RNA (4)

1 Solution D: 50 g guanidinium thiocyanate (GTC), 58.6 mL of distilled water,

3.52 mL 0.75 M trisodium citrate, 5.28 mL 10% sarkosyl NL30 Incubate at 65°C

to dissolve the GTC Store at room temperature for up to 3 mo Immediatelybefore use, add 3 5 µL of β-mercaptoethanol to 5 mL of solution D

2 2 M NaOAC, pH 4.0 Store at 4°C

3 Phenol:chloroform (4:1): water-saturated phenol, pH 4.0 chloroform (4:1) Thiscan be purchased commercially (e.g., CamLabs) Store at 4° C

3 Methods

3.1 Isolation of Mononuclear Cells from Whole Blood

1 Dilute 10–20 mL of whole blood 1:1 with ice-cold PBS

2 Carefully layer onto an equal volume of the appropriate density gradient medium,e.g., Histopaque 1077, in a 30-mL sterile tube

3 Centrifuge at 600g for 30 min at 22°C

4 Carefully collect the cellular interface using a sterile Pasteur pipet and pend the cells in 50 mL of ice-cold PBS

re-sus-5 Centrifuge at 800g for 30 min at 22°C to pellet the cells

6 Re-suspend the cells in 1 mL of ice-cold PBS and store on ice until use

3.2 Isolation of Platelets from Whole Blood (see Note 8)

1 Platelet rich plasma (PRP) is prepared from whole blood by centrifuging 10 mL

of whole blood at 600g for 10 min at 22°C (The leukocyte count of PRP is erally <0.2 × 109/L if carefully prepared.)

gen-2 Carefully aspirate the PRP and layer onto 1 mL of 38% or 40% albumin

3 Centrifuge at 1800g for 20 min at 22°C The albumin acts as a cushion ontowhich the platelets will form a lawn

4 Collect the platelet lawn and carefully resuspend in 10 mL of PBS

5 Centrifuge at 1800g for 20 min at 22°C to pellet the platelets

6 Carefully decant the supernatant and resuspend the platelet pellet in 1 mL of PBS

3.3 DNA Isolation Using the Phenol/Chloroform Method (1)

1 Add 10 mL of anticoagulated whole blood to 90 mL of ice-cold sucrose lysis

buffer, invert several times to mix and store on ice for 15 min See Note 1 for use

of smaller volumes

2 Centrifuge the samples at 1000g for 10 min at 4°C Decant the supernatant into abeaker and vigorously resuspend the pellet in 4.5 mL of TE, pH 8.0 The superna-tant is potentially infectious and should be disposed of accordingly

3 Lyse the cell pellet by adding 10 mL of nuclear lysis buffer and gently rotatingthe sample on a mechanical rotator at approx 250 rpm until a clear viscous solu-tion is obtained Samples can be frozen at this stage for processing at a later date

4 Add 5 mL of buffer-saturated phenol/chloroform and mix on a mechanical

rota-tor for 10 min (see Notes 2 and 3).

5 Centrifuge the samples at 1000g for 5 min at 20°C to separate the phases fully remove the upper aqueous phase to a clean tube using a wide-bore pipet.Avoid aspirating any of the lower phase which contains phenol/chloroform anddenatured proteins

Care-6 Add 5 mL of chloroform to the aqueous phase and mix on a mechanical rotatorfor 5 min

7 Centrifuge the samples at 1000g for 5 min at 20°C to separate the phases fully remove the upper aqueous phase to a clean tube using a wide-bore pipet

Care-8 Precipitate the DNA by adding 21/2 vol of ethanol and gently inverting the tube.Collect the DNA onto a sealed sterile glass Pasteur pipet, briefly rinse in 70%ethanol and re-suspend in 50 µL of sterile, distilled water or TE, pH 8.0 TheDNA samples are stored at 4°C for 2–3 d to allow the DNA to go into solutionand then stored at –20°C

3.4 DNA Isolation Using the Salt-Precipitation Method (3)

1 Add 5 mL of whole blood to 20 mL of sucrose lysis buffer and mix by invertingseveral times

2 Centrifuge the samples at 1000g for 10 min at 4°C Decant the supernatant into a

beaker and add 5 mL of TKM 1 to the pellet Centrifuge the samples at 1000g for

10 min at 4°C

3 Decant the supernatant and resuspend the pellet in 0.8 mL of TKM 2 Transferthe content into 2-mL labeled Eppendorf Add 25 µL of 20% SDS to the tube andgently mix Incubate at 57°C in a waterbath/heating block for 30 min

4 Transfer the Eppendorf(s) into a benchtop rack and add 300 µL of 6 M NaCl, mix

thor-oughly Centrifuge in a benchtop microfuge at maximum speed (13,000 rpm) for 10 min

5 Transfer the supernatant into a clean 2-mL Eppendorf and add an equal volume

of chloroform and mix by inversion Centrifuge at 7,000 rpm for 5 min

6 Transfer the supernatant into a sterile 20-mL tube and place on ice Add an equalvolume of ice-cold absolute ethanol and gently invert to precipitate the DNA.Collect the DNA onto a sealed sterile glass Pasteur pipette, rinse briefly in 70%ethanol and re-suspend in 500 µL of sterile, distilled water or TE, pH 8.0 TheDNA samples are stored at 4°C for 2–3 d to allow the DNA to go into solutionand then stored at –20°C

3.5 Isolation of Total Cellular RNA (4) (see Notes 5 and 6)

1 Reusupend the mononuclear pellet from 10 mL of whole blood in 1.6 mL ofsolution D and vortex vigorously for 30 s Split into 2 × 800 µL aliquots in 2-mL

Eppendorfs (see Note 7).

2 To each tube add 80 µL of 2 M NaOAc, pH 4.0 and vortex briefly to mix.

3 Add 800 µL water-saturated phenol, pH 4.0 to each tube and vortex briefly to mix

4 Add 160 µL chloroform:isoamyl alcohol (24:1) to each tube and vortex briefly tomix Leave one ice for 15 min

5 Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min Collect the upperaqueous layer and transfer to a clean 2-mL Eppendorfs.

6 Add 800 µL of propan-2-ol to each tube and place at –20°C for 1–2 h (or night)

over-7 Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min Remove the natant and resuspend each pellet in 300 µL of solution D Pool pairs of tubes

super-8 Add 600 µL of propan-2-ol and place at –20°C for 1–2 h

9 Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min Remove the natant and resuspend the pellet in 400 µL of TE, pH 8.0

super-10 Check the yield and quality of the RNA by electrophoresing 5–10 µL in a 1.5%agarose gel in 1X Trios-Borate-EDTA (TBE) The 28S and 18S (and sometimesthe 5S) ribosomal bands should be clearly visible

For long-term storage, the RNA should be precipitated by adding 21⁄2vol ofethanol and 1/10 vol of 3 M NaOAc and then placed at –70°C

4 Notes

1 For packed cells, i.e., samples in which the plasma has been removed, 5 mL ofpacked cells is added to 45 mL of cell lysis buffer Whole blood samples whichhave been frozen are thawed at 37°C and the tubes rinsed thoroughly with celllysis buffer For small volumes (100–200 µL) of blood, packed cells, leukocytebuffy coats, and so on, the cells should be lysed in 1.5 mL of cell lysis buffer,centrifuged in a benchtop microfuge at 12,000 rpm for 30 s, the supernatant care-fully aspirated, and the lysis step repeated The pellet should be resuspended is100–200µL of TE, pH 8.0 and the nuclei lysed by adding twice the volume ofnuclear lysis buffer and vortexing the sample for 10–20 s The lysed cells arethen extracted once with 200 µL of phenol-chloroform and once with 200 µL ofchloroform and the DNA precipitated by adding either 21/2 volume of ice-coldethanol or an equal volume of prop-2-ol The DNA is collected by centrifugation at12,00 rpm in a microfuge for 30 s, washed carefully in 1 mL of 70% ethanol,allowed to dry at room temperature and then re-suspended in 50 µL of TE, pH 8.0

2 Isoamyl alcohol (IAA) is commonly included in preparations of chloroform as a24:1 (v/v) mixture to prevent foaming However, this is not a problem using thetechniques described and is not routinely included

3 Phenol is extremely toxic and should be handled with care in a fume hood, ing suitable protective clothing including goggles

wear-4 All solutions for RNA isolation must be treated with DEPC carbonate) Tris-containing solutions cannot be treated in the manner and should

(diethylpyro-be made using DEPC-treated water and then autoclaved

5 To make DEPC-treated water add 1 mL of DEPC to 1 L of distilled water bate at 37°C overnight and then autoclave Store at room temperature

Incu-6 DEPC is a potential carcinogen and must be handled with care

7 Cells may be lysed in Solution D and then stored at –70°C until convenient We havecombined with subsequent processing by the RNeasy™ kit, with great success

8 Various commercial density gradient media, e.g., Nycodenz, which permit theisolation of a highly purified platelet fraction from whole blood.

References

1 Bell, G I., Karman, J H., and Rutter, W J (1981) Polymorphic DNA region

adjacent to the 5' end of the human inuslin gene Proc Natl Acad Sci USA 78(9),

5759–5763

2 Maniatis, T., Fritsch E F., and Sambrook, J (1989) Molecular Cloning: A

Labo-ratory Manual, 2nd ed Cold Spring Harbor LaboLabo-ratory Press, Cold Spring

Har-bor, NY

3 Lahiri, D K and Nurnberger, J I (1991) A rapid non-enzymatic method for the

preparation of HMW DNA from blood for RFLP studies Nucleic Acids Res 19, 5444.

4 Chomczynski, P and Sacchi, N (1987) Single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162,

156–159

From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols

Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ

PCR involves a complex series of chemical reactions in which a definedsequence of DNA (or a cDNA) is enzymatically amplified, resulting in theaccumulation of many millions of copies of the original sequence In eachcycle, both strands are templates for the generation of two new duplex mol-ecules Repeated cycles of amplification, therefore, lead to a theoretical dou-bling of the number of target molecules in each round of synthesis Each cycle

of amplification is initiated by melting of the double-stranded DNA template(denaturation) to generate a single-stranded DNA template, followed by theannealing of short synthetic oligonucleotide primers that flank the sequence ofinterest Finally, an in vitro DNA synthesis extension using a thermostableDNA polymerase, copies the DNA template, generating a double-strandedDNA molecule For the amplification of RNA sequences, an additional step isrequired to convert the single-stranded RNA into a single-stranded cDNA,which is then suitable for amplification by PCR

1 Genomic DNA: 100–500 ng (see Note 1).

2 20 mM dNTP mix: A mix of dNTPs (dATP, dCTP, dGTP, and dTTP) is prepared in

distilled water and stored at –40°C The initial concentration of each dNTP in thereaction mix (assuming a 100 µL reaction) is 200 µM, i.e., 20 mM diluted 100-fold.

Ready prepared stocks of dNTPs are available from many sources and are convenientand reliable These must be diluted and mixed before use to generate a master mix

3 10X PCR buffer (see Note 2): There are a numbers of buffers for use in PCR and

many of these are dependent upon the specific thermostable DNA polymerase.Buffers are generally made up as a 10X stock, divided into 500-µL aliquots andstored at –20°C to –70°C until required Buffers are frequently supplied by themanufacturer with a specific DNA polymerase In general we find that one of thefollowing buffers generates excellent results:

Buffer 1 (10X): 100 mM Tris-HCl, pH 8.4, 500 mM KCl, 1% Triton X-100 Buffer 2 (10X): 160 mM (NH4)2SO4, 670 mM Tris-HCl, pH 8.8, 0.1% Tween-20

4 25 mM MgCl2: Many 10X buffers include MgCl2at a concentration of 15 mM (final concentration 1.5 mM) However, in some cases adjustment of the Mg con-

centration may increase the yield and/or specificity of the final PCR product We

therefore routinely add the Mg separately (see Notes 3 and 4).

5 Synthetic oligonucleotide primers: Diluted in water to 100 pmoles/µL (see Note 5).

6 Thermostable DNA polymerase diluted to 1 U/µL in 1X PCR buffer (see Note 6).

8 DMSO: DMSO to a final concentration of 10% is included in some amplification

reactions (see Note 7).

7 Mineral oil (Sigma)

9 Thermal cycler, e.g., Perkin-Elmer

2.2 Reverse Transcription and Amplification of RNA

2.2.1 Reverse Transcription of RNA

Molecular biology grade reagents and RNAse-free sterile disposableplasticware should be used wherever possible All chemicals/reagents should

be reserved for RNA use only All solutions should be DEPC-treated or madefrom DEPC-treated solutions Disposable gloves should be worn at all times

1 5–50 ng of total cellular RNA or X-Xng of mRNA (see Note 8).

2 Reverse transcription primer: 50 pmoles/µL This may be the downstream cation primer for the subsequent PCR or an Oligo (dT)15-17 primer (see Note 9).

amplifi-3 20 mM dNTPs (dATP, dCTP, dGTP, dTTP): Make up at a 20 mM stock solution

and store at –20°C to –70°C

4 10X PCR buffer: 100 mM Tris-HCl, pH 8.4, 500 mM KCl (see Note 10).

5 25 mM MgCl2

6 AMV (Avian myeloblastosis virus) reverse transcriptase: 25 U/µL

7 RNAse inhibitor: RNAsin 5 U/µL (Promega)

8 DEPC-treated water (see Note 11).

3 Methods

3.1 PCR Amplification of DNA

Many PCR reactions require optimization to ensure efficient and specificamplification of the target DNA sequence The following protocol provides astarting point that has proven successful in our hands for the amplification ofmany target sequences Modifications and optimization of the reactions arecovered in the notes section

In a 0.5-mL sterile, thin-walled PCR tube, combine:

1 10 µL of 10X PCR buffer

2 6 µL 25 mM MgCl2 This results in a final concentration of magnesium in a 100 µL

PCR reaction volume of 1.5 mM If a magnesium-containing PCR buffer is used,

this step should be omitted

3 1 µL of 20 mM dNTP stock: Final concentration of each dNTP is 200 µM.

4 2 µL of primer mix containing 100 pmoles of each amplification primer (see

Note 5).

5 XX µL of sterile, distilled water The precise volume is dependent upon the actualconcentration of the DNA, dNTPs and primers but the final volume of the entirereaction will be 100 µL The water should be added prior to step 6.

6 Place on the surface of a UV transilluminator for 10 min

7 Add X µL of DNA (~500 ng–1 µg) The precise volume is dependent on the DNAconcentration

8 Add 1–2 U of a thermostable DNA polymerase

9 Mix gently and spin briefly in a microcentrifuge to pellet any drops of fluid thatmay be adherent to the sides of the tube

10 Overlay with 100 µL of mineral oil and place in a programmable heating blockpreheated to 94°C In some PCR blocks, the use of a heated lid prevents evapora-tion and therefore eliminates the need for oil

11 Amplification: An initial denaturation is usually carried out at 94°C for 5 min

(see Note 12) This ensures that all the double-stranded DNA is single-stranded

prior to amplification The reaction is then cooled as rapidly as possible to theannealing temperature, which allows the single-stranded DNA template to bind

to the amplification primers The annealing temperature is dependent on manyvariables, including the length and sequence of the amplification primers, but auseful starting point if using 20-mer amplification primers is 50°C The tubesshould remain at this temperature for 5 s The temperature is then raised to theextension temperature usually 72–74°C The extension times depend on the size

of the fragment being amplified, but a useful starting point is to assume an

exten-sion rate of 1 kb/min In practice, much shorter extenexten-sion times can be used out any problem Finally the temperature is raised to 94°C to denature the double-stranded DNA The cycle is then repeated Most templates will require 30–40rounds of amplification.

with-12 Following amplification, the PCR reaction is carefully removed to a clean tube,taking care not to carry over any oil 5–10 µL of each amplification is then run on

an agarose gel to check the efficiency and specificity of the reaction

3.2 Amplification of Total Cellular RNA

The amplification of RNA is preceded by a reverse transcription step, whichgenerates a single-stranded DNA molecule that is complementary to the origi-nal RNA In the subsequent amplification reaction, the single-stranded DNA isconverted to a double-stranded DNA molecule by the action of the DNA poly-merase

3.2.1 cDNA Synthesis

In a sterile, 0.5-mL Eppendorf, combine:

1 100–500 ng of total cellular RNA The precise volume will depend on the centration of the RNA

con-2 20 pmoles of the downstream amplification primer or 50 pmoles of an oligo(dT)15–17primer If the downstream amplification primer is used as a primer forthe reverse transcription, it should be purified before use either by HPLC or by

gel electrophoresis (see Note 13).

3 200 µmoles of each dNTP (dATP, dCTP, dGTP, dTTP)

4 2 µL of 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl).

5 6 µL of 25 mM MgCl2

6 Incubate at 65°C for 10 min, then place on ice and add 20 U of RNAsin and 20 U

of AMV reverse transcriptase

7 DEPC-treated sterile water to 20 µL

8 Incubate at 20°C for 10 min, 42°C for 60 min, and finally at 95°C for 10 min toinactivate the reverse transcriptase

9 Following reverse transcription, samples are stored on ice until required or zen at –80°C

fro-3.2.2 PCR Amplification of Single-Stranded cDNA

Combine the following in a 0.5-mL thin-walled, sterile Eppendorf:

1 2–20µL of the reverse transcription reaction The precise volume depends on thefrequency of the particular RNA species that is being amplified For rare RNAspecies, the entire reverse transcription reaction may be required

2 8 µL of 10X PCR buffer (see Note 14).

3 2 µL 25 mM MgCl2

4 100 pmoles of each amplification primer

5 Sterile water to 100 µL.

6 The amplification reactions are heated to 100°C for 5 min and then allowed tocool to 20°C for 2 min to allow the primer to anneal to the single-stranded DNA.This is conveniently performed in the PCR block 2.5 U of a thermostable DNApolymerase is then added to each tube, e.g., Amplitaq, and the samples overlaidwith 100 µK of light mineral oil The samples are then incubated at 70°C for 10min to allow the generation of a double-stranded DNA molecule, followed by 40cycles of PCR

7 Following amplification 8–10 µL of each reaction is run on agarose gel to checkthe efficiency and specificity of the reaction

4 Notes

1 a PCR can be used to amplify DNA from many sources, e.g., genomic DNA,plasmid DNA, bacterial DNA, and so on For the amplification of cloned DNA aslittle as 10–20 ng may be sufficient As a general guide, the amount of DNA used

as a template should be increased as the complexity of the source increases

b The fidelity of an amplification reaction is dependent on a number of ables including the DNA concentration Any errors introduced in the first fewcycles will be amplified in subsequent cycles The fraction of the final prod-uct that contains a mutation is inversely proportional to the number of initialDNA molecules If the number of starting DNA molecules is small, it may beadvisable to use a DNA polymerase with proofreading ability

2 a When all else fails, the following buffer is often useful: 670 mM Tris-HCl pH8.8, 166 mM (NH4)2SO4, 67 mM MgCl2, 1.5 mg/mL bovine serum albumin and

100 mM b-mercaptoethanol Store the inorganic solution at room temperatureand add the BSA and b-ME immediately before use The high concentration of

Mg2+ often results in nonspecific products

b Many manufacturers include gelatin (final concentration 0.01%) or Triton

X-100 (final concentration 0.1%) in the buffer to stabilize the enzyme duringthermal cycling Inclusion of such components may increase the efficiency ofthe amplification reaction

3 a dNTPs bind to Mg2+in a 1:1 molar ratio, i.e., 0.8 mM dNTPs will bind 0.8

mM Mg2+ Therefore, in an amplification mix containing a final Mg2+centration of 1.5 mM, only 0.7 mM Mg2+(1.5–0.8 mM) is “free” and active as

con-a cofcon-actor for the DNA polymercon-ase If the concentrcon-ation of dNTPs is creased, e.g., in long-range PCR, then the amount of bound Mg2+increasesand the functionally active free Mg2+decreases In such cases there may beinsufficient Mg2+for the enzyme to function It is important, therefore, that ifthe concentration of dNTPs in a reaction mix is increased, the concentration

in-of Mg2+ is also increased

b To prevent misincorporation of nucleotides, it is important the all four dNTPsare present in equal concentration

4 The concentration of Mg2+in the PCR mix is one of the key variables

affect-ing both yield and specificity High Mg2+concentrations lead to nonspecific

amplification by stabilizing priming at incorrect template sites Furthermore,high Mg2+ concentrations may also stabilize double-stranded DNA thereforereducing the yield of the final PCR product Low Mg2+concentrations mayalso affect the yield of the final PCR product as Mg2+is required as a cofactorfor the DNA polymerase (see Note 3).

5 a Primer design has become greatly simplified by development of various puter programmes, e.g., Lasergene and Oligo 4 and in our experience very fewprimer pairs fail to work having been designed using such programmes

com-b The length of a primer can vary between 17–26mers For PCR from complexsources such as genomic DNA, 20–26mers allow amplification of specificsequences In contrast if the template is simpler then shorter primers, e.g.,17mers may suffice There are a number of rules that should be observedwhen designing primers, e.g., GC content, avoiding 3' complementary ends,and so on For a guide to the design of primers, the reader is referred to one ofthe many excellent PCR primers available

c The concentration of primers in the PCR mix should be similar unless metric PCR is being performed Primers are usually received resuspended indistilled water and their concentration is given If primers are received freezedried they should be resuspended in 100–500 µL of distilled water If theconcentration of the primers is unknown this can be calculated by measuringtheir optical density (OD) at 260nm (1 OD260= 40 µg of single-stranded oli-gonucleotide)

asym-We routinely mix relevant primer pairs, such that 2 µL of the mix contain 100pmoles of each primer and scale up the mix so that we have enough of theprimer mix for 50 or 100 amplifications, e.g.:

Primer A: Concentration = 400 µM (= 400 pmol/µL)

Primer B: Concentration = 500 µM (= 500 pmol/µL)

Therefore, a mix comprising 0.25 µL of Primer A + 0.2 µL of Primer B + 1.55 µL

of distilled water will contain 100 pmol of each primer in a final volume of 2 µL

To make sufficient primer mix for 100 separate amplifications these volumes aremultiplied by 100, i.e., 25 µL of Primer A + 20 µL of Primer B + 155 µL of water

6 Thermostable DNA polymerases isolated from a variety of bacteria are now able for use in the PCR process In many cases the choice is dictated by availabil-ity and price However, some DNA polymerases, e.g., “Vent” and “Pfu,” have a3'–5' proofreading exonuclease activity that can significantly reduce the number

avail-of misincorporation errors that occur during PCR Such enzymes are, extremelyuseful if the number of starting template molecules are small or if the final PCRproduct is to be cloned and expressed Enzymes with 3'–5' proofreading exonu-clease activity should not be used for allele-specific amplification and similarlythey cannot be used (without modification of the final PCR product) if the ampli-fied product is to be cloned using a T-vector, e.g., “TA Cloning System”(Invitrogen) as the product contains no overhangs

7 DMSO to a final concentration of 10% may increase the efficiency and/or ficity of some DNA polymerases However, there are some reports that DMSO

speci-may be inhibitory to certain DNA polymerases and it is not, therefore, routinelyincluded in our amplification reactions.

8 The quality of the RNA is crucial in RT-PCR Techniques for isolating RNAfrom a wide variety of cells are now available Total cellular RNA is adequate forthe vast majority of experiments and rarely do we find any necessity to isolatemRNA species

9 Reverse transcription of RNA using the downstream amplification primer usedsubsequently for the PCR is routinely used and generates excellent results for awide variety of templates However, Oligo(dT)15–17(50 pmol) can also be usedwith similar results

10 We do not routinely use an RT-specific buffer but instead the KCl-based PCRbuffer For most situations this appears to work satisfactorily For some experi-ments it may be necessary to use an RT-specific buffer Such buffers are fre-quently supplied with the reverse transcriptase enzyme

11 Diethyl pyrocarbonate (DEPC) is a suspected carcinogen and should be used withcare in a fume hood DEPC should not be used directly in Tris-based solutions

but such solutions can be made with DEPC-treated water (see Chapter 1).

12 The efficiency and specificity of the PCR can often be improved by the use of a

“hot start.” A “hot start” involves omitting a key component from the PCR mix,e.g., DNA polymerase, and denaturing the sample at 94–100°C for 5 minutes.The missing component is then added directly to the tube and then overlaid withmineral oil or the heated lid lowered

13 Oligonucleotides for use in reverse transcription experiments are purified beforeuse This is most easily achieved by HPLC purification but an alternativeapproach involves electrophoresis in 20% denaturing polyacrylamide gels fol-lowed by elution 50 cm × 20 cm × 1 mm 20% polyacrylamide gels are cast using

72 g urea, 75 mL of 40% acrylamide (19:1 acrylamide/bisacrylamide) and merized with 150 µL of TEMED and 150 µL of 25% ammonium persulphate (gelvolume = 150 mL) A 5-well comb with 200 µL wells is used Gels are pre-run at

poly-60 W for 30 min before use Approximately 0.02 µmol of crude oligonucleotide

is mixed with 0.5 vol of loading buffer (95% formamide, 20 mM EDTA, 0.05%

bromophenol blue, 0.05% xylene cyanole) in a final volume of 100–150 µL,incubated at 100°C for 5 min, and loaded onto the gel Gels are run at 60 Wconstant power in 1X TBE until the bromophenol blue dye front has reached theend of the gel The plates are then separated and the DNA visualized by UVshadowing Bands of the correct size are excised, placed in a 2-mL Eppendorf,and crushed with a pipet tip 1.5 mL of water is added and the DNA eluted intothe distilled water by vigorous shaking (in an orbital shaker at 250 rpm) at 37°Cfor 24 h The DNA is then placed in dialysis tubing (SpectraPor No 6 dialysistubing) and dialyzed against distilled water for 48 h at 4° C, changing the waterevery 12 h The oligonucleotide is then freeze-dried, re-suspended in 50–100 µL

of distilled water, and quantitated by measuring its OD260 From the formula:

(OD260× dilution × 40/309 × mer) × 103

where “mer” is the length of the oligonucleotide and “dilution” the dilution of theoligonucleotide prior to measuring its OD260, the concentration of the oligonucle-otide can be established So, for example, if 20 µL of a 26-mer oligonucleotide isdiluted to 1 mL and its OD260is 0.449 then the concentration of the oligonucle-otide is 118 µM (0.118 mM), which equals 118 pmol/µL.

14 This is usually the same buffer used in the reverse transcription reaction, unless areverse transcriptase specific buffer has been used

Suggested Reading

Erlich, H A (1989) PCR Technology: Principles and Applications for DNA

Amplifi-cation Stockton Press, London.

McPherson, M J., Hames, B D., and Taylor, G R (1995) PCR 2: A Practical Approach.

IRL, Oxford, UK

White, B A (1993) PCR Protocols Humana Press, Totowa, NJ.

From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols

Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ

In general, the most important part of sequencing a PCR product is the ity of the DNA template and in particular ensuring that it is free from anycontaminating amplification primers or dNTPs Residual amplification prim-ers that may be present in a sequencing reaction can act as sequencing primers,making the final sequence unreadable A number of approaches have beentaken to try to overcome this problem Initially, PCR products were cloned andthen sequenced However, cloning is time-consuming even with the use ofPCR-specific cloning vectors, and furthermore, a single clone providessequence data on only a single DNA molecule Subsequent developmentsallowed “direct” sequencing of amplified DNA following purification of thePCR products There are countless physical methods for purifying a PCR prod-uct prior to sequencing, e.g., GeneClean™, Sephadex G50 chromatography,agarose gel electrophoresis, and so on To some extent the success of thesemethods is dependent on the size of the PCR product GeneClean, for example

qual-is ideal for purifying PCR products that are greater than 500 bp in length but qual-is

of little value for products that are less than 400 bp

An entirely different approach to generating single-stranded DNA involvesthe use of asymmetric PCR in which one of the amplification primers is present

in limited amounts, such that one primer is exhausted and linear amplificationthen takes place or alternatively the PCR product forms the basis of a secondPCR in which only one amplification primer is present

Two different protocols are covered in this chapter The first involves labeling a “nested” sequencing primer, i.e., one that is internal to the amplifica-tion primers, with either [γ32P]-dATP or [γ33P]-dATP and which is then used tosequence a purified PCR template The advantage of this technique is that itallows sequence data to be derived up to the sequencing primer, which may be ofvalue when sequencing short PCR products The second method involves [α-

radio-35S]-dATP rather [γ-32P]-dATP and nested sequencing primers However, in trast to the first method, the primer is not radio-labelled but instead [35S]-dATP

con-is incorporated during the sequencing reaction Direct solid-phase sequencing ofPCR products using biotinylated primers is covered in a separate chapter

2 Materials

All reagents are molecular grade of equivalent Many of the sequencingreagents are available commercially in kit form, e.g., Sequenase v2 (AmershamLife Sciences) Although the method described is for sequencing using alabeled oligonucleotide sequencing primer, methods are also included for theuse of a nonlabeled primer and the incorporation of the radioisotope during thesequencing reaction

2.1 Direct Sequencing of PCR Generated Templates

Using 5'-Labeled Oligonucleotide Primers

Direct sequencing of PCR products using a 5'-labeled oligonucleotide primerinvolves five separate steps:

1 5'-labeling of the oligonucleotide primer

2 Purification of the PCR product to provide a template for sequencing

3 Annealing of the primer to the template

4 Extension-termination reactions

5 Casting, running, fixing, and drying the sequencing gel

2.1.1 5'-Labeling of Oligonucleotide Sequencing Primers

Using T4 Polynucleotide Kinase

Synthetic oligonucleotides for use as sequencing primers can be labeled attheir 5'-terminus using T4 polynucleotide kinase:

1 Synthetic oligonucleotide primer: 25 pmol/µL in water (see Note 1).

2 10 X T4 polynucleotide kinase (T4 PNK) buffer: 700 mM Tris-HCl, pH 7.6, 100

mM MgCl2, 5 mM dithiothreitol (DTT) Store at –20°C

3 Bovine serum albumin at 10 mg/ mL

4 [γ32P]-dATP (5000 Ci/mmol) (see Note 2) Available from a variety of sources,

e.g., Amersham Life Sciences

5 T4 polynucleotide kinase: 10 U/µL

6 Distilled water

2.2 Purification of PCR Products for Sequencing

A variety of methods are available for the purification of PCR productsprior to sequencing The methods listed below have all been used with greatsuccess For products greater than 500 bp, GeneClean (or similar) is the method

of choice

2.2.1 Purification of PCR Products by Sephadex G50 Chromatography

1 Sephadex G50 (Pharmacia): Preswollen and stored at 4°C in TE, pH 8.0

2 TE, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

3 1-mL syringe in which the plunger has been removed and the end of the syringeplugged with glass wool

4 100 µL of crude PCR product

2.2.2 Purification of PCR Products Using GeneClean II

DNA is bound to a glass matrix, washed, and then eluted Any small DNAfragments, e.g., primers, remain bound to the glass matrix and only the largerDNA fragments are eluted

1 GeneClean II Kit (BIO 101 Inc, La Jolla, California) GeneClean contains “Glass

Milk”–a DNA binding matrix, 6 M sodium iodide (NaI) and a concentrated “wash

solution.” Before use, the wash solution must be reconstituted by mixing 14 mL

of the provided concentrate with 280 mL of distilled water and then adding 310 mL

of 100% ethanol This should be stored at –20°C

2.2.3 Purification of PCR Products by Agarose Gel ElectrophoresisPCR products are run in low gelling (melting) temperature agarose Theband of interest is excised and purified either by GeneClean II or by a “freeze-thaw” method

1 Low gelling temperature agarose (available from many laboratories)

2 1X Tris-Acetate-EDTA (1X TAE): 0.04 M Tris-acetate, 0.001 M EDTA (see Note 3).

3 TE, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

4 TE-saturated phenol (see Note 4).

5 Chloroform

6 Ethidium bromide 0.5 µg/mL (see Note 5).

7 7.5 M Ammonium acetate.

8 Absolute ethanol

9 Sterile, distilled water

10 Sucrose loading buffer: 40% sucrose (w/v), 0.25% bromophenol blue (w/v), and0.25% xylene cyanole (w/v)

2.2.4 Purification of PCR Products by Selective Precipitation

In this method large DNA fragments are selectively precipitated and thenpurified, whereas free primers and dNTPs remain in solution

1 7.5 M Sodium acetate.

2 Absolute ethanol

3 70% ethanol

4 Sterile, distilled water

2.3 Annealing Primer to Template and Sequencing

of the PCR Product

Sequencing of the purified PCR template involves annealing the 5'-labeledprimer to the purified single-stranded DNA template and then performing theextension-termination reactions

1 Sequencing primer (concentration 1 pmol/µL) labeled at its 5'-end with [γ32dATP

P]-2 5X Reaction buffer: 200 mM Tris-HCl pH 7.5, 100 mM MgCl2, 250 mM NaCl.

Store at –20°C Thaw before use and keep on ice

3 Purified DNA template–at least 150 ng

4 0.1 M Dithiothreitol (DTT) Store at –20°C Thaw before use and keep on ice

5 T7 DNA Polymerase (e.g., Sequenase v2.0; 14 U/µL) Store at –20°C Do notremove from the freezer—aliquots should be removed as required directly intothe prechilled sequencing master mix

6 Enzyme dilution buffer: 100 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.5 mg/mL

bovine serum albumin Store at –20°C Thaw before use and keep on ice

7 Termination mixes (see Note 6) Store at –20°C Thaw before use and keep onice until required

ddGTP termination mix: 50 mM NaCl, 80 µM dGTP, 80 µM dATP, 80 µM

dCTP, 80 µM dTTP, and 8 µM ddGTP ddATP termination mix: 50 mM NaCl, 80 µM dGTP, 80 µM dATP, 80 µM

dCTP, 80 µM dTTP, and 8 µM ddATP ddTTP termination mix: 50 mM NaCl, 80 µM dGTP, 80 µM dATP, 80 µM

dCTP, 80 µM dTTP, and 8 µM ddTTP ddCTP termination mix: 50 mM NaCl, 80 µM dGTP, 80 µM dATP, 80 µM

6 10X Tris-Borate-EDTA (TBE).

7 Duck-billed tips for loading the gel

8 Sequencing plates (40/50 cm × 20 cm)

9 Silanizing solution, e.g., dimethylchlorosilane (Sigma)

10 2 L of 5% methanol/5% acetic acid in water

11 Whatman 3MM paper

12 Autoradiograph film, e.g., Kodak

3 Methods

3.1 Direct Sequencing of PCR Generated Templates

Using 5'-Labeled Oligonucleotide Primers

3.1.1 5'-Labeling of Oligonucleotide Sequencing Primers

Using T4 Polynucleotide Kinase

Twenty-five pmol of sequencing primer is labeled in a final volume of 25 µL.This is sufficient for 25 separate sequencing reactions

1 Place a 1.5-mL sterile Eppendorf on ice and add: X µL of water [the exact ume will vary depending upon the concentration of the oligonucleotide but thefinal volume of the reaction should be 25 µL], 25 pmol of sequencing primer in avolume of X µL, 2.5 µL of 10X T4-PNK buffer, 1 µL of bovine serum albumin,7.5µL (75 µCi) of [γ32P]-dATP Add 1 µL (10 U) of T4-polynucleotide kinase

vol-2 Incubate at 37°C for 60 min and then at 85°C for 15 min to inactivate the T4PNK

3 Use 1 µL (1 pmol) of the labeled oligonucleotide per template The labeled primermay be safely stored at –20°C for 7–10 d but its usable life-span is limited by theshort half-life of the [32P]-dATP Purification of the labeled template away fromthe unincorporated radio-nucleotide is unnecessary

3.2 Purification of PCR Products for Sequencing

3.2.1 Purification of PCR Products by Sephadex G50 Chromatography

1 Plug the end of a 1-mL disposable plastic syringe with glass wool and pack withSephadex G50 (Pharmacia-LKB Ltd.) preswollen with TE, pH 8.0

2 Place the syringe into a 15-mL tube (e.g., Falcon) and centrifuge at 200g for 3 min.

3 Apply the crude PCR product to the top of the syringe

4 Centrifuge at 200g for 3 min.

5 Collect the eluate which contains the purified PCR product

6 Evaporate under vacuum to reduce the volume, e.g., in a Speedivac Final ume should be ~7 µL

vol-3.2.2 Purification of PCR Products for Sequencing Using GeneClean

II (see Note 7)

1 Add 3 vol of 7 M NaI to the crude PCR product This is generally 540 µL of NaIfor 80 µL of the PCR product

2 Vortex the Glass Milk until all the contents are in suspension and then add10 µL

to the NaI-PCR solution and place on ice for 20 min The samples should beinverted several times during this period to keep the Glass Milk in suspension

3 Pellet the Glass Milk by spinning in a microcentrifuge at 12,000 rpm for 5 s

4 Carefully aspirate the supernatant and discard

5 Add 500 µL of cold wash solution to the Glass Milk and vortex to resuspend theGlass Milk Efficient re-suspension of the Glass Milk is extremely important

6 Pellet the Glass Milk by spinning in a microcentrifuge at 12,000 rpm for 5 s.Carefully aspirate the supernatant and discard

7 Repeat the wash/spin step twice After the second spin, spin the tube again topellet any remaining wash solution, carefully aspirate and discard

8 Add 20 µL of distilled water to the Glass Milk and resuspend the pellet

9 Incubate at 55°C for 20 min Spin at 12,000 rpm for 30 s Carefully remove thesupernatant to a clean tube A second elution can be performed but in practise theadditional yield of DNA is very small

3.2.3 Purification of PCR Products by Agarose Gel Electrophoresis

1 Pour a 10 × 5 × 0.5 cm, 1% low gelling temperature agarose gel in 1X TAEcontaining ethidium bromide 0.5 µg/mL Use a comb or well former that createswells capable of holding 50–60 µL

2 Submerge the gel in chilled 1X TAE

3 Add 10 µL of sucrose loading buffer to 100 µL PCR product and load into thewells of the gel

4 Electrophoresis is performed at no more than 5–10 V/cm otherwise the gel willmelt!

Electrophoresis can be performed at room temperature but the sis times can be shortened either by running the gel at 4°C or by placing theelectrophoresis apparatus onto a bed of ice

electrophore-5 When the xylene cyanole dye has migrated to the end of the gel, place the gelonto the surface of a UV transilluminator, the surface of which has been previ-ously covered with Saran Wrap™ Visualize and excise the band of interest underlow power UV light and place into a sterile Eppendorf

6 To denature the gel matrix and release the DNA, place the gel slice at –80°C for

5 min and then at 37°C for 10 min Repeat this step once

7 Centrifuge the gel slice in a benchtop microfuge at 12,000 rpm for 20 min

8 Collect the supernatant, extract once with an equal volume of TE-saturated nol and once with equal volume of chloroform

phe-9 Precipitate the DNA by adding 1/10 vol of 7.5 M ammonium acetate, 2 1/2 vol of

ethanol, and place the sample at –80°C for 20–30 min in a microfuge

10 Collect the DNA by centrifugation at 12,000 rpm for 10 min, wash the DNApellet in 400 µL 70% ethanol and finally resuspend in 20 µL of sterile distilledwater Use 7 µL for the sequencing reaction