Biochemistry, 4th Edition P40 potx

So, if a single bacterial cell harboring a recombinant DNA molmol-ecule in the form of a plasmid grows and multiplies on a petri plate to form a colony, the plasmids within the millions

Further Reading 353

Luger, C., et al., 1997 Crystal structure of the nucleosome core particle

at 2.8 Å resolution Nature 389:251–260.

Rhodes, D., 1997 The nucleosome core all wrapped up Nature 389:

231–233.

Chromosome Structure

Pienta, K J., and Coffey, D S., 1984 A structural analysis of the role of

the nuclear matrix and DNA loops in the organization of the nucleus

and chromosomes In Cook, P R., and Laskey, R A., eds., Higher

order structure in the nucleus Journal of Cell Science Supplement

1:123–135.

Sumner, A T., 2003 Chromosomes: Organization and Function Malden, MA:

Blackwell Science.

Tremethick, D J., 2007 Higher-order structures of chromatin: The

elu-sive 30 nm fiber Cell 128:651-654.

Telomeres

Axelrod, N., 1996 Of telomeres and tumors Nature Medicine 2:158–159.

Feng, J., Funk, W D., Wang, S-S., Weinrich, S L., et al., 1995 The RNA

component of human telomerase Science 269:1236–1241.

Chemical Synthesis of Genes

Ferretti, L., Karnik, S S., Khorana, H G., Nassal, M., and Oprian, D D.,

1986 Total synthesis of a gene for bovine rhodopsin Proceedings of the

National Academy of Sciences U.S.A 83:599–603.

Higher-Order RNA Structure

Ban, N., et al., 2000 The complete atomic structure of the large

riboso-mal subunit at 2.4 Å resolution Science 289:905–920.

Gray, M W., and Cedergren, R., eds., 1993 The new age of RNA The

FASEB Journal 7:4–239 A collection of articles emphasizing the new

appreciation for RNA in protein synthesis, in evolution, and as a catalyst.

Holbrook, S R., 2005 RNA structure: The long and the short of it

Cur-rent Opinion in Structural Biology 15:302–308.

Klosterman, P S., et al., 2005 Three-dimensional motifs from the SCOR, structural classification of RNA database: Extruded strands, base

triples, tetraloops, and U-turns Nucleic Acids Research 32:2342–2352.

Nilsen, T W., 2007 RNA 1997–2007: A remarkable decade of discovery.

Molecular Cell 28:715–720.

Scala/Art Resource, NY

Cloning and Creation

of Chimeric Genes

In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged In turn, these technologies led to the construction of DNA molecules com-posed of nucleotide sequences taken from different sources The products of these

investi-gation in molecular biology and genetics, and a new field was born—recombinant

DNA technology Genetic engineering is the application of this technology to the

manipulation of genes These advances were made possible by methods for

amplifi-cation of any particular DNA segment, regardless of source, within bacterial host

cells Or, in the language of recombinant DNA technology, the cloning of virtually

any DNA sequence became feasible

In classical biology, a clone is a population of identical organisms derived from a

sin-gle parental organism For example, the members of a colony of bacterial cells that arise from a single cell on a petri plate are a clone Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original mol-ecule or cell So, if a single bacterial cell harboring a recombinant DNA molmol-ecule

in the form of a plasmid grows and multiplies on a petri plate to form a colony, the plasmids within the millions of cells in the bacterial colony represent a clone of the original DNA molecule, and these molecules can be isolated and studied Further-more, if the cloned DNA molecule is a gene (or part of a gene)—that is, it encodes

a functional product—a new avenue to isolating and studying this product has opened Recombinant DNA methodology offers exciting new vistas in biochemistry

Plasmids Are Very Useful in Cloning Genes

Plasmidsare naturally occurring, circular, extrachromosomal DNA molecules (see

Chapter 11) Natural strains of the common colon bacterium Escherichia coli isolated

from various sources contain diverse plasmids Often these plasmids carry genes specifying novel metabolic activities that are advantageous to the host bacterium These activities range from catabolism of unusual organic substances to metabolic functions that endow the host cells with resistance to antibiotics, heavy metals, or

bacteriophages Plasmids that are able to perpetuate themselves in E coli, the

bac-terium favored by bacterial geneticists and molecular biologists, are the workhorses

The Chimera of Arezzo, of Etruscan origin and

proba-bly from the fifth century B.C., was found near Arezzo,

Italy, in 1553 Chimeric animals existed only in the

imagination of the ancients But the ability to create

chimeric DNA molecules is a very real technology

that has opened up a whole new field of scientific

investigation.

…how many vain chimeras have you created?…

Go and take your place with the seekers after

gold.

Leonardo da Vinci

The Notebooks (1508–1518), Volume II, Chapter 25

KEY QUESTIONS

12.1 What Does It Mean “To Clone”?

12.2 What Is a DNA Library?

12.3 Can the Cloned Genes in Libraries Be

Expressed?

12.4 What Is the Polymerase Chain Reaction

(PCR)?

12.5 How Is RNA Interference Used to Reveal the

Function of Genes?

12.6 Is It Possible to Make Directed Changes

in the Heredity of an Organism?

ESSENTIAL QUESTIONS

Using techniques for the manipulation of nucleic acids in the laboratory, scientists can join together different DNA segments from different sources Such manmade products are called recombinant DNA molecules, and the use of such molecules to alter the genetics of organisms is termed genetic engineering

What are the methods that scientists use to create recombinant DNA mole-cules; can scientists create genes from recombinant DNA molemole-cules; and can scientists modify the heredity of an organism using recombinant DNA?

Create your own study path for

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1 The advent of molecular biology, like that of most scientific disciplines, generated a jargon all its own Learning new fields often requires gaining familiarity with a new vocabulary We will soon see that

many words—vector, amplification, and insert are but a few examples—have been bent into new

mean-ings to describe the marvels of molecular biology.

12.1 What Does It Mean “To Clone”? 355

of recombinant DNA technology Because restriction endonuclease digestion of

plasmids can generate fragments with overlapping or “sticky” ends, artificial

plas-mids can be constructed by ligating different fragments together Such artificial

plasmids were among the earliest recombinant DNA molecules These recombinant

molecules can be autonomously replicated, and hence propagated, in suitable

bac-terial host cells, provided they still possess a site signaling where DNA replication

can begin (a so-called origin of replication or ori sequence).

Plasmids as Cloning Vectors The idea arose that “foreign” DNA sequences could

be inserted into artificial plasmids and that these foreign sequences would be

car-ried into E coli and propagated as part of the plasmid That is, these plasmids could

serve as cloning vectors to carry genes (The word vector is used here in the sense of

“a vehicle or carrier.”) Plasmids useful as cloning vectors possess three common

fea-tures: a replicator, a selectable marker, and a cloning site (Figure 12.1) A replicator

is an origin of replication, or ori The selectable marker is typically a gene conferring

resistance to an antibiotic Only cells containing the cloning vector will grow in the

presence of the antibiotic Therefore, growth on antibiotic-containing media

“se-lects for” plasmid-containing cells Typically, the cloning site is a sequence of

nu-cleotides representing one or more restriction endonuclease cleavage sites Cloning

sites are located where the insertion of foreign DNA neither disrupts the plasmid’s

ability to replicate nor inactivates essential markers

Virtually Any DNA Sequence Can Be Cloned Nuclease cleavage at a restriction site

opens, or linearizes, the circular plasmid so that a foreign DNA fragment can be

in-serted The ends of this linearized plasmid are joined to the ends of the fragment so

that the circle is closed again, creating a recombinant plasmid (Figure 12.2)

Recom-binant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus

inserted DNA elements (called inserts) Such hybrid molecules are also called chimeric

constructs or chimeric plasmids (The term chimera is borrowed from mythology and

refers to a beast composed of the body and head of a lion, the heads of a goat and a

snake, and the wings of a bat.) The presence of foreign DNA sequences does not

ad-versely affect replication of the plasmid, so chimeric plasmids can be propagated in

bacteria just like the original plasmid Bacteria often harbor several hundred copies of

common cloning vectors per cell Hence, large amounts of a cloned DNA sequence

4

H ind

III

Ec oRV Nh eI Bam

HI

EagI NruI BspMI

BsmI

StyI Ava

I

Bsp

MII

Pvu

Nd

el

3

4

1

2

ori

Bal

I

Afl

III

PpaI

PstI

Pvu

I

Sca

I

Ssp

I

Sph

I

Aa tII

pBR322 (4363 bases)

FIGURE 12.1 One of the first widely used cloning vec-tors, the plasmid pBR322 This 4363-bp plasmid contains

an ori and genes for resistance to the drugs ampicillin (ampr) and tetracycline (tetr ) The locations of restriction endonuclease cleavage sites are indicated.

can be recovered from bacterial cultures The enormous power of recombinant DNA

technology stems in part from the fact that virtually any DNA sequence can be selectively

cloned and amplified in this manner DNA sequences that are difficult to clone include

in-verted repeats, origins of replication, centromeres, and telomeres The only practical limitation is the size of the foreign DNA segment: Most plasmids with inserts larger than about 10 kbp are not replicated efficiently However, bacteriophages such as

be inserted into the bacteriophage genome Such recombinant phage DNA

Construction of Chimeric Plasmids Creation of chimeric plasmids requires join-ing the ends of the foreign DNA insert to the ends of a linearized plasmid This ligation is facilitated if the ends of the plasmid and the insert have complementary, single-stranded overhangs Then these ends can base-pair with one another, an-nealing the two molecules together One way to generate such ends is to cleave the DNA with restriction enzymes that make staggered cuts; many such restriction endo-nucleases are available (see Table 10.2) For example, if the sequence to be inserted

1

2

3

T T

A A C A A T G C

G

Cut with EcoRI

T T

A A C G

G A A

G A A

G

C T T A A

DNA ligase

Cut with EcoRI

Anneal ends of vector and foreign DNA

Seal gaps in chimeric plasmid with DNA ligase

ACTIVE FIGURE 12.2 An EcoRI

restric-tion fragment of foreign DNA can be inserted into a

plasmid having an EcoRI cloning site by (1) cutting the

plasmid at this site with EcoRI, (2) annealing the

lin-earized plasmid with the EcoRI foreign DNA fragment,

and (3) sealing the nicks with DNA ligase Test yourself

on the concepts in this figure at www.cengage.com/

login.

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BiochemistryInteractive to explore the

construction of chimeric plasmids.

12.1 What Does It Mean “To Clone”? 357

is an EcoRI fragment and the plasmid is cut with EcoRI, the single-stranded sticky

ends of the two DNAs can anneal (Figure 12.2) The interruptions in the

sugar–phosphate backbone of DNA can then be sealed with DNA ligase to yield a

covalently closed, circular chimeric plasmid DNA ligase is an enzyme that

that any pair of EcoRI sticky ends can anneal with each other So, plasmid molecules

can reanneal with themselves, as can the foreign DNA restriction fragments These

DNAs can be eliminated by selection schemes designed to identify only those

bac-teria containing chimeric plasmids

Blunt-end ligation is an alternative method for joining different DNAs The most

widely used DNA ligase, bacteriophage T4 DNA ligase, is an ATP-dependent enzyme

that can even ligate two DNA fragments whose ends lack overhangs (blunt-ended

DNAs) Many restriction endonucleases cut double-stranded DNA so that blunt

ends are formed

A great number of variations on these basic themes have emerged For example,

short synthetic DNA duplexes whose nucleotide sequence consists of little more

than a restriction site can be blunt-end ligated onto any DNA These short DNAs are

known as linkers Cleavage of the ligated DNA with the restriction enzyme then

leaves tailor-made sticky ends useful in cloning reactions (Figure 12.3) Similarly,

many vectors contain a polylinker cloning site, a short region of DNA sequence

bearing numerous restriction sites

Promoters and Directional Cloning Note that the strategies discussed thus far

create hybrids in which the orientation of the DNA insert within the chimera is

ran-dom Sometimes it is desirable to insert the DNA in a particular orientation For

ex-ample, an experimenter might wish to insert a particular DNA (a gene) in a vector

so that its gene product is synthesized To do this, the DNA must be placed

down-stream from a promoter A promoter is a nucleotide sequence lying updown-stream of a

gene The promoter controls expression of the gene RNA polymerase molecules

bind specifically at promoters and initiate transcription of adjacent genes, copying

template DNA into RNA products One way to insert DNA so that it will be properly

oriented with respect to the promoter is to create DNA molecules whose ends have

different overhangs Ligation of such molecules into the plasmid vector can only

take place in one orientation to give directional cloning (Figure 12.4).

(a)

P

P

P

P

P

P

DNA ligase

EcoRI

(b) A vector cloning site containing multiple restriction sites,

a so-called polylinker.

EcoRI BamHI SalI

AccI HincII

PstI SalI AccI HincII

ANIMATED FIGURE 12.3 (a) The use of

linkers to create tailor-made ends on cloning fragments Note that the ligation reaction can add multiple linkers

on each end of the blunt-ended DNA EcoRI digestion

removes all but the terminal one, leaving the desired

5-overhangs (b) Cloning vectors often have polylinkers

consisting of a multiple array of restriction sites at their cloning sites, so restriction fragments generated by a variety of endonucleases can be incorporated into the vector Note that the polylinker is engineered not only

to have multiple restriction sites but also to have an uninterrupted sequence of codons, so this region of the vector has the potential for translation into protein (see Figure 12.15) (Adapted from Figure 1.14.2 in Greenwich, D., and

Brent, R., 2003 UNIT 1.14 Introduction to Vectors Derived from Filamentous Phages, in Current Protocols in Molecular Biology,

Ausubel, F M., Brent, R., Kingston, R E., Moore, D D., Seidman, J G., Smith, J A., and Struhl, K., eds New York: John Wiley and Sons.)See this figure animated at www.cengage.com/login.

Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore blunt-end ligation.

Biologically Functional Chimeric Plasmids The first biologically functional chimeric DNA molecules constructed in vitro were assembled from parts of differ-ent plasmids in 1973 by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert

Helling These plasmids were used to transform recipient E coli cells (transformation

means the uptake and replication of exogenous DNA by a recipient cell) To facili-tate transformation, the bacterial cells were rendered somewhat permeable to DNA

could be selected by their resistance to certain antibiotics (Figure 12.5) Conse-quently, the chimeric plasmids must have been biologically functional in at least two

EcoRI SacI KpnI SmaI BamHI XbaI SalI

SphI HindIII

pUC19

EcoRI SacI KpnI

pUC19

PstI

P

3'

3'

5' 5'

P

BamHI

HindIII

Small fragment discarded

Digest with

HindIII and BamHI

Isolate large fragment by electrophoresis or chromatography

Target DNA Digest with

HindIII and BamHI

P

P

Target DNA anneals with plasmid vector

in only one orientation Seal with T4 DNA ligase.

EcoRI SacI KpnI SmaI BamHI

HindIII

pUC19

Large fragment

PstI

SphI

XbaI SmaI

SalI

ANIMATED FIGURE 12.4 Directional

cloning DNA molecules whose ends have different

over-hangs can be used to form chimeric constructs in which

the foreign DNA can enter the plasmid in only one

ori-entation The foreign DNA and the plasmid are digested

with the same two enzymes pUC stands for universal

cloning plasmid See this figure animated at www

.cengage.com/login.

12.1 What Does It Mean “To Clone”? 359

aspects: They replicated stably within their hosts, and they expressed the drug

re-sistance markers they carried

In general, plasmids used as cloning vectors are engineered to be small (2.5 kbp

to about 10 kbp in size) so that the size of the insert DNA can be maximized These

plasmids have only a single origin of replication, so the time necessary for complete

replication depends on the size of the plasmid Under selective pressure in a

grow-ing culture of bacteria, overly large plasmids are prone to delete any nonessential

“genes,” such as any foreign inserts Such deletion would thwart the purpose of

B am

HI

2

4

5

6

3

1

Ec in

dIII

Ec oRV

SalI

am

pr tetr

ori

pBR322

(4363 bases)

Ava

I

Sal

I

Pvu

I

Ps

tI

Pvu

II

am p

r

BamHI restriction fragment of

DNA to be cloned is inserted

into the BamHI site of tetr.

ampr gene remains intact.

Chimeric plasmid

Suspend 20 ng plasmid DNA + 10 7

E.coli cells in CaCl2 solution.

Plate bacteria on ampicillin media.

42⬚C, 2 min

37 ⬚C, overnight

Ampicillin-containing medium

Only ampicillin-resistant

(ampr ) bacterial colonies grow.

Using velvet-covered disc, bacterial colonies are lifted from surface of agar

amprplate and pressed briefly to surface

of plate containing tetracycline media.

37⬚C, overnight

Only tetr colonies appear;

tets colonies can be

recovered from ampr plate

by comparing two plates.

tetr gene is inactivated by the insertion of DNA fragment.

ampr gene remains intact.

Tetracycline-containing medium

A plasmid with genes for

ampicillin resistance (ampr ) and

tetracycline resistance (tetr).

A BamHI restriction site is

located within the tetr gene.

ACTIVE FIGURE 12.5 A typical bacterial transformation experiment Here the plasmid pBR322

is the cloning vector (1) Cleavage of pBR322 with BamHI, followed by (2) annealing and ligation of inserts

generated by BamHI cleavage of some foreign DNA, (3) creates a chimeric plasmid (4) The chimeric plasmid is

then used to transform Ca2-treated heat-shocked E coli cells, and the bacterial sample is plated on a petri

plate (5) Following incubation of the petri plate overnight at 37°C, (6) colonies of ampr bacteria are evident.

(7) Replica plating of these bacteria on plates of tetracycline-containing media (8) reveals which colonies are

tetrand which are tetracycline sensitive (tets) Only the tets colonies possess plasmids with foreign DNA inserts.

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most cloning experiments The useful upper limit on cloned inserts in plasmids is about 10 kbp Many eukaryotic genes exceed this size

Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms

Shuttle vectors are plasmids capable of propagating and transferring (“shuttling”) genes between two different organisms, one of which is typically a prokaryote

(E coli) and the other a eukaryote (for example, yeast) Shuttle vectors must have

unique origins of replication for each cell type as well as different markers for se-lection of transformed host cells harboring the vector (Figure 12.6) Shuttle vectors have the advantage that eukaryotic genes can be cloned in bacterial hosts, yet the expression of these genes can be analyzed in appropriate eukaryotic backgrounds

Artificial Chromosomes Can Be Created from Recombinant DNA

DNA molecules 2 megabase pairs in length have been successfully propagated in

yeast by creating yeast artificial chromosomes or YACs Furthermore, such YACs

have been transferred into transgenic mice for the analysis of large genes or multi-genic DNA sequences in vivo, that is, within the living animal For these large DNAs

to be replicated in the yeast cell, YAC constructs must include not only an origin of

replication (known in yeast terminology as an autonomously replicating sequence or

ARS) but also a centromere and telomeres Recall that centromeres provide the site

for attachment of the chromosome to the spindle during mitosis and meiosis, and telomeres are nucleotide sequences defining the ends of chromosomes Telomeres are essential for proper replication of the chromosome

A DNA library is a set of cloned fragments that collectively represent the genes of a specific organism Particular genes can be isolated from DNA libraries, much as books can be obtained from conventional libraries The secret is knowing where and how to look

Insert DNA

amp r

Polycloning site

Yeast

LEU2+

Yeast origin of replication

Bacterial origin

of replication

Transform

LEU– yeast

E.coli

Yeast cell

Plasmids can be shuttled between

E.coli and yeast

Shuttle vector

Transform

E.coli

ANIMATED FIGURE 12.6 A typical shuttle vector LEU2is a gene in the yeast pathway for

leucine biosynthesis The recipient yeast cells are LEU2(defective in this gene) and thus require leucine for

growth LEU2yeast cells transformed with this shuttle vector can be selected on medium lacking any leucine

supplement See this figure animated at www.cengage.com/login.

12.2 What Is a DNA Library? 361

Genomic Libraries Are Prepared from the Total DNA in an Organism

Any particular gene constitutes only a small part of an organism’s genome For

gene is 10 kbp, then the gene represents less than 0.001% of the total nuclear DNA

It is impractical to attempt to recover such rare sequences directly from isolated

nu-clear DNA because of the overwhelming amount of extraneous DNA sequences

In-stead, a genomic library is prepared by isolating total DNA from the organism,

di-gesting it into fragments of suitable size, and cloning the fragments into an

appropriate vector This approach is called shotgun cloning because the strategy has no

way of targeting a particular gene but instead seeks to clone all the genes of the

or-ganism at one time The intent is that at least one recombinant clone will contain at

least part of the gene of interest Usually, the isolated DNA is only partially digested

by the chosen restriction endonuclease so that not every restriction site is cleaved in

every DNA molecule Then, even if the gene of interest contains a susceptible

re-striction site, some intact genes might still be found in the digest Genomic libraries

have been prepared from thousands of different species

Many clones must be created to be confident that the genomic library contains

the gene of interest The probability, P, that some number of clones, N, contains a

particular fragment representing a fraction, f, of the genome is

Thus,

N

For example, if the library consists of 10-kbp fragments of the E coli genome (4640 kbp

total), more than 2000 individual clones must be screened to have a 99% probability

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

Combinatorial Libraries

Specific recognition and binding of other molecules is a defining

characteristic of any protein or nucleic acid Often, target ligands

of a particular protein are unknown, or in other instances, a

unique ligand for a known protein may be sought in the hope of

blocking the activity of the protein or otherwise perturbing its

func-tion Or, the hybridization of nucleic acids with each other

accord-ing to base-pairaccord-ing rules, as an act of specific recognition, can be

exploited to isolate or identify pairing partners Combinatorial

li-brariesare the products of strategies to facilitate the identification

and characterization of macromolecules (proteins, DNA, RNA)

that interact with small-molecule ligands or with other

macromole-cules Unlike genomic libraries, combinatorial libraries consist of

synthetic oligomers Arrays of synthetic oligonucleotides printed as

tiny dots on miniature solid supports are known as DNA chips (See

the section titled “DNA Microarrays (Gene Chips) Are Arrays of

Dif-ferent Oligonucleotides Immobilized on a Chip.”)

Specifically, combinatorial libraries contain very large numbers

of chemically synthesized molecules (such as peptides or

oligonu-cleotides) with randomized sequences or structures Such libraries

are designed and constructed with the hope that one molecule

among a vast number will be recognized as a ligand by the protein

(or nucleic acid) of interest If so, perhaps that molecule will be

useful in a pharmaceutical application For instance, the synthetic

oligomer may serve as a drug to treat a disease involving the

pro-tein to which it binds

An example of this strategy is the preparation of a synthetic

combinatorial library of hexapeptides The maximum number of

sequence combinations for hexapeptides is 206, or 64,000,000

One approach to simplify preparation and screening possibilities

for such a library is to specify the first two amino acids in the hexa-peptide while the next four are randomly chosen In this approach,

400 libraries (202) are synthesized, each of which is unique in terms

of the amino acids at positions 1 and 2 but random at the other four positions (as in AAXXXX, ACXXXX, ADXXXX, etc.), so each

of the 400 libraries contains 204, or 160,000, different sequence combinations Screening these libraries with the protein of interest reveals which of the 400 libraries contains a ligand with high affin-ity Then, this library is expanded systematically by specifying the first three amino acids (knowing from the chosen 1-of-400 libraries which amino acids are best as the first two); only 20 synthetic libraries (each containing 203, or 8000, hexapeptides) are made here (one for each third-position possibility, the remaining three positions being randomized) Selection for ligand binding, again with the protein of interest, reveals the best of these 20, and this particular library is then varied systematically at the fourth posi-tion, creating 20 more libraries (each containing 202, or 400, hexapeptides) This cycle of synthesis, screening, and selection is repeated until all six positions in the hexapeptide are optimized

to create the best ligand for the protein A variation on this basic strategy using synthetic oligonucleotides rather than peptides identified a unique 15-mer (sequence GGTTGGTGTGGTTGG)

with high affinity (KD 2.7 nM ) toward thrombin, a serine

pro-tease in the blood coagulation pathway Thrombin is a major target for the pharmacological prevention of clot formation in coronary thrombosis

From Cortese, R., 1996 Combinatorial Libraries: Synthesis, Screening and Ap-plication Potential Berlin: Walter de Gruyter.

(P  0.99) of finding a particular fragment Since ƒ  10/4640  0.0022 and P  0.99,

kbp human genome, N would equal almost 1.4 million if the cloned fragments

aver-aged 10 kbp in size The need for cloning vectors capable of carrying very large DNA inserts becomes obvious from these numbers

Libraries Can Be Screened for the Presence of Specific Genes

A common method of screening genomic libraries is to carry out a colony

hybridization experiment In a typical experiment, host bacteria containing a plasmid-based library are plated out on a petri dish and allowed to grow overnight

to form colonies (Figure 12.7) A replica of the bacterial colonies is then obtained

by overlaying the plate with a flexible, absorbent disc The disc is removed, treated with alkali to dissociate bound DNA duplexes into single-stranded DNA, dried, and placed in a sealed bag with labeled probe (see the Critical Developments in Bio-chemistry box on page 364) If the probe DNA is duplex DNA, it must be denatured

by heating at 70°C The probe and target DNA complementary sequences must be

in a single-stranded form if they are to hybridize with one another Any DNA se-quences complementary to probe DNA will be revealed by autoradiography of the absorbent disc Bacterial colonies containing clones bearing target DNA are identi-fied on the film and can be recovered from the master plate

Probes for Southern Hybridization Can Be Prepared

in a Variety of Ways

Clearly, specific probes are essential reagents if the goal is to identify a particular gene against a background of innumerable DNA sequences Usually, the probes that are used to screen libraries are nucleotide sequences that are complementary to some part of the target gene Making useful probes requires some information about the gene’s nucleotide sequence Sometimes such information is available Alterna-tively, if the amino acid sequence of the protein encoded by the gene is known, it is possible to work backward through the genetic code to the DNA sequence (Figure

12.8) Because the genetic code is degenerate (that is, several codons may specify the

same amino acid; see Chapter 30), probes designed by this approach are usually

degenerate oligonucleotides about 17 to 50 residues long (such oligonucleotides are so-called 17- to 50-mers) The oligonucleotides are synthesized so that different bases are incorporated at sites where degeneracies occur in the codons The final prepa-ration thus consists of a mixture of equal-length oligonucleotides whose sequences vary to accommodate the degeneracies Presumably, one oligonucleotide sequence

in the mixture will hybridize with the target gene These oligonucleotide probes are

at least 17-mers because shorter degenerate oligonucleotides might hybridize with sequences unrelated to the target sequence

A piece of DNA from the corresponding gene in a related organism can also be used as a probe in screening a library for a particular gene Such probes are termed

1

3 2

4

5

Master plate of

bacteria colonies

Replicate onto

absorbent disc.

Denatured

DNA bound

to absorbent

disc

Radioactive probe will hybridize with its complementary DNA

Autoradiograph film

Place disc in sealable plastic bag with solution

of labeled DNA probe.

Treat with NaOH;

neutralize, dry.

Wash disc,

prepare

auto-radiograph,

and compare

with master

plate.

Darkening identifies colonies containing the DNA desired

ACTIVE FIGURE 12.7 Screening a genomic library by colony hybridization Host bacteria transformed with a plasmid-based genomic library are plated on a petri plate and incubated overnight to allow bacterial colonies to form A replica of the colonies is obtained by overlaying the plate with a flexible disc

composed of absorbent material (such as nitrocellulose or nylon) (1) Nitrocellulose strongly binds nucleic

acids; single-stranded nucleic acids are bound more tightly Once the disc has taken up an impression of the

bacterial colonies, it is removed and the petri plate is set aside and saved The disc is treated with 2 M NaOH,

neutralized, and dried (2) NaOH both lyses any bacteria (or phage particles) and dissociates the DNA strands.

When the disc is dried, the DNA strands become immobilized on the filter The dried disc is placed in a sealable

plastic bag, and a solution containing heat-denatured (single-stranded), labeled probe is added (3) The bag is

incubated to allow annealing of the probe DNA to any target DNA sequences that might be present on the

disc The filter is then washed, dried, and placed on a piece of X-ray film to obtain an autoradiogram (4) The position of any spots on the X-ray film reveals where the labeled probe has hybridized with target DNA (5).

The location of these spots can be used to recover the genomic clone from the bacteria on the original petri

plate Test yourself on the concepts in this figure at www.cengage.com/login.