Section I Gene library and screening

 Representative gene libraries Gene libraries made from genomic DNA are called genomic libraries and those made from complementary DNA are known as cDNA libraries..  Genomic DNA For m

Section I Gene library and

screening

I 1 Genomic libraries

Gene libraries and

screening

 Representative gene libraries Gene libraries made from genomic DNA are called genomic libraries and those made from

complementary DNA are known as cDNA libraries The latter lack nontranscribed genomic sequences(repetitive sequences, etc.) Good gene libraries are representative of the starting material and have not lost certain sequences due to cloning artifacts.

 Size of library A gene library must contain a certain number of recombinant for there to be a high probability of it containing

any particular sequence This value can be calculated if the

genome size and the average size of the insert in the vector are known.

 Genomic DNA For making libraries, genomic DNA, usually

prepared by protease digestion and phase extraction, is

fragmented randomly by physical shearing or restriction enzyme digestion to give a size range appropriate for the chosen vector Often combination of restriction enzymes are used to partially digest the DNA.

 Vectors Plasmids, λ pahge, cosmid, BAC or yeast artificial

chromosome vectors can be used to construct genomic libraries, the choice depending on the genome size The upper size limit of these vectors is about 10,23,45,350 and 1000 kb respectively The genomic DNA fragments are ligated to the prepared vector

I 2 cDNA libraries

 mRNA isolation, purification and

fractionation

mRNA can be readily isolated from lysed

eukaryotic cells by adding magnetic beads which have oligo(dT) covalently attached The mRNA

binds to the oligo(dT) via its poly(A) tail and thus

be isolated from the solution The integrity of an mRNA preparation can be checked by translation in

a wheat germ extract or reticulocyte lysate and

then visualizing the translation products by

polyacrylaminde gel electrophoresis Integrity can also be studied using gel electrophoresis, which

allows the mRNA to be size fractionated by

recovering chosen regions of the gel lane Specific sequences can be removed from the mRNA by

hybridization.

Gene libraries and

screening

deoxyribounucletides to 3’-end Synthesis can be detected

by trace labeling 3’-Tailing of the first strand cDNA using terminal transferase makes full-length second strand

synthesis easier Reverse transcriptase or Klenow enzyme can extend a primer [e.g.oligo(dG)] annealed to a

homopolymeric tail [e.g.oligo(dC)] to synthesize second

strand cDNA.

 Treatment of cDNA ends To avoid blunt end ligation of

cDNA to vector, linkers are usually added to the cDNA after the ends have been repaired(blunted) using a single

strand-specific nuclease followed by Klenow enzyme The cDNA may also be methylated to keep it from being

digested when the added linkers are cleaved by a

restriction enzyme Adaper molecules can be used as an alternative to linkers.

 Ligation to vector The vector is usually dephosphorylated using alkaline phosphatase to prevent self-ligation, and so promote the formation of recombinant molecules Plasmid

or phage vectors can be used to make cDNA libraries, but the phage λgt11 is preferred for the cnstruction of

expression libraries.

Fig 1 cDNA cloning (a) First and second strand synthesis; (b) end preparation and linker addition to duplex cDNA.

 Screening Screening to isolate one particular clone

from a gene library routinely involves using a nucleic acid probe for hybridization The probe will bind to its complementary sequence allowing the required clone

to be identified.

 Clony and plaque hybridization A copy of the position

of colonies or plaques on a petri is made on the surface

of a membrane, which is then incubated in a solution of labeled probe After hybridization and washing, the

location of the bound label is determined The group of colonies/plaques to which the label has bound is

diluted and re-plated in subsequent rounds of

screening until an individual clone is obtained.

 Expression screening One-sixth of the clones in a cDNA

expression libraries will produce their encoded polypeptide as

a fusion protein with the vector-encodeed β-galactosidase

Antibidies to the desired protein can be used to screen the

library in a process similar to plaque hybridization to obtain particular clone.

 Hybrid arrest and release cDNA clones can be hybridized to mRNA preparations to prevent, or arrest, the subsequent

translation of some mRNA species Alternatively, those mRNA hybridized to the cDNA clones can be purified, released from the cDNA and translated to identify the polypeptides encoded.

 Chromosome walking This is the repeated screening of

genomic libraries to obtain overlapping clones and hence build

up a collection of clones covering part of a chromosome It

involves using the ends of the inserts as probes for subquent rounds of screening Chromosome jumping is a similar process

Fig 1 Screening by plaque hybridization

Fig 2 Chromosome walking