Section I Gene library and screening

Molecular Biology
Section I Gene library and screening
Molecular Biology
Gene libraries and screening
I1 Genomic libraries I2
cDNA libraries
I3 Screening procedures
Molecular Biology
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 βgalactosidase. 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.
Molecular Biology
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Gene libraries and screening
I2
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.
Molecular Biology
Fig. 1. cDNA cloning. (a) First and second strand synthesis; (b) end preparation and linker addition to duplex cDNA.
Molecular Biology
Molecular Biology
Gene libraries and screening
I1 Genomic libraries
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 molecule using T4 DNA ligase.
Molecular Biology Synthesis of cDNA In the first synthesis, reverse transcriptase is used to make a cDNA copy of the mRNA by exetending a primer, usually oligo(dT), by the addition of 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.