Question

Describe the basic process to clone a cDNA vs. genomic library using PCR technique

Answer 1

Meaning of cDNA Library:

A cDNA library is defined as a collection of cDNA fragments, each of which has been cloned into a separate vector molecule.

Principle of cDNA Library:

In the case of cDNA libraries we produce DNA copies of the RNA sequences (usually the mRNA) of an organism and clone them. It is called a cDNA library because all the DNA in dis library is complementary to mRNA and is produced by the reverse transcription of the latter.

Much of eukaryotic DNA consists of repetitive sequences that are not transcribed into mRNA and the sequences are not repre­sented in a cDNA library. It must be noted that prokaryotes and lower eukaryotes do not con­tain introns, and preparation of cDNA is generally unnecessary for these organisms. Hence, cDNA libraries are produced only from higher eukaryotes.

Vectors used in the Construction of cDNA Library:

Both the bacterial and bacteriophage DNA are used as vectors in the construction of the cDNA library.

It is sometimes the case that we wish to clone a particular cDNA for which we already have some sequence data but wif a particular emphasis on the integrity of the 5′ or 3′ ends. RACE techniques (Rapid Amplification of cDNA Ends) are available for dis. The RACE methods are divided into 3’RACE and 5’RACE, accord­ing to which end of the cDNA we are in­terested in.

a) 3’RACE:

In this type of RACE, reverse transcriptase synthesis of a first DNA strand is carried out using a modified oligo-dT primer. This primer comprises a stretch of unique adaptor se­quence followed by an oligo-dT stretch. The first strand synthesis is followed by a second-strand synthesis using a primer internal to the coding sequence of interest.

dis is followed by PCR using

(me) The same internal primer and ‘

(ii) The adaptor sequence (me.e., omitting the oligo-dT). Although in theory, it should be possible to use a simple oligo- dT primer throughout instead of the adaptor-oligo-dT and adaptor combi­nation, the low melting temperature for an oligo-dT primer may interfere wif the subsequent rounds of PCR.

(b) 5’RACE:

In this type of RACE first cDNA strand is synthesized with reverse transcriptase and a primer from within the coding sequence. Unincor­porated primer is removed and the cDNA strands are tailed with oligo-dA. A second cDNA strand is then synthe­sized with an adaptor-oligo-dT primer.

The resulting double-stranded mol­ecules are then subject to PCR using

(i) A primer nested within the coding region and

(ii) The adaptor sequence. A nested primer is used in the final PCR to improve specificity. The adap­tor sequence is used in the PCR be­cause of the low melting temperature of a simple oligo-dT primer, as in 3’RACE above.

Advantages of cDNA Library:

A cDNA library has two additional advantages. First, it is enriched wif fragments from ac­tively transcribed genes. Second, introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to pro­duce a eukaryotic protein in bacteria because most bacteria have no means of removing the introns.

Genomic library:

A genomic library contains all the sequences present in the genome of an organism.

Steps in Genomic Library Construction:

Construction of genomic library involves following steps:

(а) Isolation of target DNA:

Genomic libraries can be constructed by isolation of com­plete DNA from bacteria, virus, plants and animals. In eukaryotes, high molecular weight DNA is isolated by CTAB or SDS methods. The isolated DNA is tan purified by caesium chloride and other methods.

(b) Restriction Fragments:

Fragmentation can be done by mechanical shearing or us­ing suitable restriction enzymes. Partial digestion is essential to procure proper size DNA frag­ments. Therefore, treatment times and concentration of enzyme is very important for a desirable result.

(c) Cloning the fragments in vector:

The restricted digested DNA sample is electrophoresed and subjected to. Target DNA fragments are identified by hybridization with probes and tan cloned in suitable vectors like lambda or cosmid vectors and maintained as library.

(d) Screening of Genomic library:

Genomic library can be screened for clones by hy­bridization with probe, western blotting to detect protein product and also screening of protein activity.

Screening by Colony Hybridization:

Principally this screening technique involves hybridization between labelled DNA probe and a target DNA sequence. Therefore, a target gene sequence can be accurately identified.

The essential steps are described as follows:

(i) The initial step in screening a library—a colony of host cells are plated onto solid medium containing antibiotics. The presence of antibiotics in the medium ensures the growth of only transformed cells due to its antibiotic resistance gene in their plasmid.

(ii) When a discrete colony is formed on the plate, it is tan transferred onto nitrocellu­lose or nylon membrane commonly referred as solid matrix. The exact position of cell colony on the plate is maintained on the matrix.

(iii) Once the cells are attached to a solid matrix (nitrocellulose paper) cells are lysed, deproteinized, and released DNA is denatured by the alkaline reagent.

(iv) The labeled DNA probe is mixed in the next step, to facilitate hybridization between target DNA and DNA probe. The unbound probe is washed off from the matrix

(v) Matrix is tan subjected to autoradiography to determine the location of cells con­taining hybridized DNA.

(vi) Development of dark spot on the X-ray film indicates the presence of target DNA (gene). The dark spot on X-ray film corresponds to the cell colony on the master plate. Once transformed cell colony is identified, it is then sub-cultured and main­tained as it carries cloned DNA of interest.

Plaque Hybridization:

A genomic library is screened by infecting phage viruses containing inserted DNA on a lawn of bacteria.

The steps are described as follows:

(i) A lawn of bacterial cultures is initiated on teh agar plate.

(ii) Several thousands of phage particles containing inserted DNA are spread on teh lawn of bacteria.

(iii) Phage infects bacteria and plaques are obtained. These are then transferred to ni­trocellulose paper; DNA is denatured to single strand and bound firmly onto solid matrix.