cDNA & Genomic DNA Library

Introduction:-

• A DNA library is a collection of clones of DNA designed so that there is a high probability of finding specific piece of the source DNA in the collection. 
• There are two types of DNA libraries:- 

I. GENOMIC LIBRARY: The genomic library contains DNA fragments representing entire genome of an organism. 

II. cDNA LIBRARY: The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. 

I. Genomic library

• A genomic library is a collection of bacterial clones or lysates that when taken together represent every segment of the Genome of an organism.
• The genomic Library Include sequences that are regulatory, specifying for proteins Specifying for structural RNAs and sequences without known function (Junk DNA). 
• In the eukaryotic genomic library, the gene sequences contain both the expressed regions (Exons) and non-expressed regions (introns.)

The construction of a genomic Library involves the following steps:-

• Isolation of genomic DNA from suitable tissue. 
• Fragmentation of the genomic DNA into fragments of convenient length. This can be carried out either by using the mechanical shearing or by using partial restriction digestion; the mechanical shearing generates the randomly sized fragments that exhibit great variation in the length whereas the partial dilation produces fragments of uniform size. 
• Electro phoretic separation of the fragments, the fragments are separated by agarose gel electrophoresis. The separation is based on the size of the fragments, smaller sized fragments migrate faster and to greater distances compare to larger sized fragments. The electro phoretic mobility of a fragment is inversely proportional to the logarithm of its molecular mass 
• Cloning of the selected fragments into suitable vectors The initial genomic libraries were constructed by using Lambda replacement vectors such as EMBL3 and EMBL4. The genomic libraries of higher eukaryotes are generated by using high capacity vectors such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) for the lower eukaryotes cosmids are preferred.

• The recombinant vectors containing the genomic DNA fragments are introduced into the cloning host (E. coli K-12 strain that is mutant for restriction modification and recombination) 
• After the transfer, the host cell will be screen for the presence of genomic DNA inserts, and the positive clones are collected and are arranged by using chromosome walking method. 
• Chromosome walking involves the arrangement of genomic library colonies in the specific order based on the overlapping of the fragments. The chromosome walking technique involves the identification of the homologous fragments at 5' and 3' ends by using hybridization technique with the 5' and 3' probes.

• After the complete analysis of the colony with hybridization based on the homology the colonies are arranged and stored at low temperature

Uses of Genomic Library:-

1. To determine the complete genome sequence of a given organism.
2. To improve the copy number of the genome 
3. Useful for chromosomal mapping (order of genes on a chromosome) 
4. It is the source for generation of transgenic animals through genetic engineering. 
5. Study of the functions of regulatory sequences in vitro 
6. Study of genetic mutations in various genetic diseases. 
7. Genomic library is a most useful method in Human Genome Project viii. Germplasm creation for species conservation.

II. cDNA Library.

• A collection of bacterial colonies or lysates when taken together represent every cDNA sequence obtained from the reverse transcription of mRNAs from specific tissue. A cDNA library represents only the expressive portion of the genome. 
• The cDNAs represent only the coding regions (exons) of the genes since the mRNAs will have only exons. The cDNA libraries are useful in isolating targeting genes that must be expressed in prokaryotic hosts.

The following are the steps involved in the construction of the cDNA Library:-

• Selection of tissue that has abundance of specific mRNAs.
• Isolation of total cellular RNA: As the from eukaryotic mRNAs are polyadenylated at their 3' ends it is advantage for isolation by affinity chromatography on a poly U Sepharose column or an oligo dT cellulose column. 
• Checking of the RNA integrity: Analysis the mRNAs by gel electrophoresis and make sure that the mRNA is not degraded. 

• Reverse transcription and generation of double stranded cDNA molecules 

  a) First strand synthesis: The first cDNA strand is synthesized with the aid of a reverse       transcriptase enzyme using either a poly dT primer or arbitrary primers or specific                 primers. 

b) Second strand synthesis: The second cDNA strand is synthesized by three methods.

 

I. Hairpin Primed Second Strand DNA Synthesis (Maniatis et al., 1976): In this method the first strand is synthesized using a reverse transcriptase enzyme with the help of an oligo dT primer. mRNAcDNA hybrid is treated with an alkaline solution (sodium hydroxide) to remove mRNA. The 3'end of the newly formed cDNA strand has the tendency to form a hairpin-like structure and acts as a primer. Based on this a second strand is made. The hairpin structure in double stranded cDNA strands thus formed is removed by using an enzyme called S1 nuclease to allow insertion into the cloning vector. But the use of the enzyme S1 nuclease in this method would result in the loss of some portion of the 5'end of cDNA.  

ii. The best way is to make a full-length cDNA is to modify the 3’end of the first strand. Land et al., (1981). added a poly cytidine terminal to the 3’end of the first cDNA strand using the terminal transferase enzyme. The second cDNA strand is then synthesized using an oligo dG primer.

iii. The Gubler–Hoffman method-Simple and general method for non-directional cDNA cloning. In this method the mRNA-cDNA hybrid is hydrolyzed using the enzyme RNase H. The partially hydrolyzed mRNA creates into small pieces and used as primers. These RNA fragments are utilized as primers by DNA polymerase enzyme to replace mRNA with new cDNA

c. Electro phoretic separation of cDNA 

d. Treatment of cDNA ends and ligation to vector Upon completion of cDNA synthesis, the cDNA ends are treated with homopolymer tails or linkers and inserted into the suitable vector (e.g. plasmid). 

e. Transformation into suitable host Introduction of the recombinant vectors with cDNA inserts into the host (E. coli K12) and screening for the positive clones and the positive clones are stored. 

f. λ phage replacement vectors are generally used for the construction of the cDNA libraries. Cosmids are rarely used. 

Uses of cDNA Library:-

• Storage of reduced amount of information due to the removal of noncoding regions 

• cDNA can be directly expressed in PK organisms 

• To isolate homologous genes 

• Cloning of full-length cDNA molecules for in vitro study of gene function 

• Study of the repertoire of mRNAs expressed in different cells or tissues 

Comparison of Genomic library and cDNA library:-

 Reference:- 

• Principles of Gene Manipulation: sixth edition, Sandy B Primrose, Richard M Twyman and Robert W Old. 
• https://www.sciencedirect.com/topics/agricultural-and-biologicalsciences/cdnalibraries#:~:text=A%20cDNA%20library%20is%20a,stands%20for%20' complementary'). 
• https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Bo ok%3A_Basic_Cell_and_Molecular_Biology_(Bergtrom)/15%3A_DNA_T echnologies/15.02%3A_Make_and_Screen_a_cDNA_Library 
• https://www.differencebetween.com/difference-between-dna-and-vscdna/