Basic concepts about cDNA and genomic-library

Library:- It is a convenient storage mechanism of genetic information.

1. cDNA library

2. Genomic library

1. cDNA library:- It is a collection of cloned DNA sequences that are complementary to the mRNA that was extracted from an organism or tissue.

> cDNA is developed by the process of reverse transcription.

> Reverse transcription:- Genetic information contained in mRNA is converted back into a double stranded DNA form. The enzyme responsible for this is called reverse transcriptase.

> Reverse transcriptases:- They are isolated from Retroviruses. Eg.- Moloney murine leukemia virus (MMLV). They have polymerase activity, but do not have exonuclease activity.

> Primer:- MMLV will use mRNA as a template, but requires a primer. Eukaryotic mRNA has a  3' poly A tail. Therefore we can use poly dT as a prime.

> RNAse H:- This enzyme has endonuclease activity, so create nicks in the RNA. 

> DNA polymese - I:- RNA fragments now serve as primers for DNA synthesis by E. coli Pol I. This enzyme also remove the RNA primers.

> T4 DNA ligase:- Nicks are sealed by this enzyme.

> Treat the cDNA with RNase H to remove possible 5' cap mRNA fragment remaining in cDNA duplex.

> Now treat the cDNA with S1 nuclease to remove 3' overhangs.

> Insertion of cDNA into plasmid:- 2 methods -

a. Homopolymeric tailing

b. Linker addition

a. Homopolymeric tailing:- Terminal transferase is an unusual DNA polymerase found only in prelymphocyte. 

- In the presence of a divalent cation the enzyme catalyzes the addition of 3 to 1000 dNTPs to the 3'-OH end of DNA.

i. When the nucleotide to be added is a purine, Mg2+ is the cation used.

ii. When the nucleotide to be added is a pyrimidine, Co2+ is used.

- Now treat the plasmid with PstI to ceate sticky ends and treat it with terminal transferase to add the complementary bases.

 - The utility of inserting the C-tailed cDNA insert into a G-tailed Pst I site in the vector is as follows:

i. The Pst I recognition sequence and cleavage site is:-

5' C T G C A ↓ G 3'  →→→→→  5' C T G C A 3'            5' G 3'

3' G ↑A C G T C 5'  →→→→→   3' G 5'           3' A C G T C 5'

ii. Cleavage of this site by Pst I, followed by G-tailing will produce:-

5' C T G C A 3'           5' G 3'  →→→→→  5' C T G C A (G)n 3'      5' G 3'

3' G 5'           3' A C G T C 5'  →→→→→   3' G 5'       3' (G)n A C G T C 5'

- Now anneal and ligate the cDNA into the plasmid.

b. Linker addition:-

- Linkers:- Linkers are short oligonucleotides (18 to 24 Ntds) which are typically palindromic and contain restriction endonuclease recognition sequence.

- If the ends of the cDNA fragments are blunt, then the linker can be ligated to both ends to introduce useful terminal restriction sites.

- The steps in linker addition are as follows:

i. Methylate cDNA at potential internal BamHI sites by treatment with BamHI methylase (plus S-adenosyl methionine).

ii. Ligate linkers to blunt, methylated cDNA using T4 DNA ligase.

iii. Cut linkers with BamHI restriction endonuclease.

iv. Remove linker fragments from cDNA fragments by agarose gel electrophoresis.

v.Ligate cDNA to vector DNA fragment (opened up by BamHI restriction endonuclease).

2. Genomic library:- 

> Size of some genomes and chromosomes:-

> The human genome contains approximately 50,000 unique genes within 3-4 billion base pairs of DNA, scattered about in 23 pairs of chromosomes.

> Fragmentation of genomic DNA:- Genomic DNA is too large to be incorporated into a vector and must be fragmented into desired sizes. RE enzymes are used for digestion.

> Insertion into lambda phage vectors:- 

- lambda phage is an E. coli phage with a type of icosahedral phage particle which contains the viral genome.

- During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.

It was found that internal regions of the phage genome, which were not essential to phage replication, could be removed and replaced with DNA of interest.

This hybrid DNA could be efficiently packaged, and form an infective phage.