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8. Compare and contrast a) cDNA library and genomic library b) RFLPs and STRs

Answer 1

a)

The gene cloning is a multi step process. The DNA of an organism is isolated and purified. Then it is cut with suitable restriction endonuclease enzyme to generate fragments of required length. The fragments are ligated into suitable vector (possibly plasmid). The recombinant molecules are then introduced into host cells and screened to identify the transformed cells. The total DNA fragments are transformed into a host cell to produce a gene library. The gene libraries are of two types called genomic and cDNA libraries.

The genomic library is the entire genome of an organism cut into fragments and then transformed into cells. The cDNA library is generated by isolation of specific genes. These genes are trasncribed to produce mRNA. From the mRNA, a complementary DNA (cDNA) is generated by using reverse transcriptase enzyme. The, the cDNA is fragmented and ligated into suitable vector. Further the cDNA is transformed into cells producing a cDNA library. The cDNA library produced from mRNA do not contain any noncoding and regulatory regions or junk. It contains only coding regions or exons of expressed genes. The other differences between genomic and cDNA libraries are the genomic library is larger compared to cDNA library.

The genomic library cannot be expressed in prokaryotic systems such as bacteria because of no suitable splicing mechanisms. Since, the cDNA library contains only coding regions, they are greatly expressed in prokaryotic systems. To construct the genomic library the vectors such as plasmid, cosmid, BAC and YAC etc can be used to accommodate large size DNA fragments. The cDNA lbrary contains only few coding sequences and thus vectors such as plasmids, phagemids and lambda phage can be used. The study and use of genomic library is complex process compared to cDNA library with few coding genes. The cDNA libraries are widely used in study of various genetic diseases, cancer and genetic mutations etc.

b)

The DNA sequences vary from individual to individual and these differences can be studied using techniques such as restriction fragment length polymorphism (RFLP) and short tandem repeats (STR) etc. The RFLP generates various length restriction fragments of DNA cut by different restriction endonuclease enzymes. The enzymes cut the DNA at specific sequences. The resultant DNA fragments of different length are viewed on gel as a pattern of bands, corresponding to the size of the fragments.

The STR or short tandem repeats are nucleotide repeats of 2 or more nucleotides that occur mostly in the non-coding parts or introns of the DNA. These are highly polymorphic and the amount of repetitions in a specific STR can be different from person to person. The DNA samples are analyzed by amplifying either one or more STR regions. The differences in the size of the STRs between the samples can be separated on gel based on size. Small amount of sample than RFLP is required in STRsbecause PCR can amplify the sample.

Both the techniques are used for the study of forensics, paternity issues, genetic markers of a disease, gene mapping, selection of traits in breeding.