The cloning of a gene from an organism is very costly and time consuming. If the focus is made on the cloning of a specific gene, the genomic DNA has to be isolated, purified and cut into fragments. The fragments are then separated by electrophoresis.
The fragment of interest is identified by Southern blot hybridization. Then the fragment is inserted into a cloning vector for transformation of a host cell. The transformed host cell is grown on culture medium for viable clones. Each time one proposes to clone a gene, all the aforementioned steps are repeated, which proves to be time consuming and wasteful.
Therefore, it was proposed to construct recombinant DNA of all the fragments of the genomic DNA digest and preserve in the form of a library of transformed host cells so that a gene could be retrieved from the library and cloned as and when required. There are two types of gene libraries: 1. Genomic DNA Library and 2. CDNA Library. 1. Genomic DNA Library:
In this library, all the genomic DNA fragments are represented in the form of recombinant DNA transformed host cell clones. This method is also called shotgun cloning.
Preparation of DNA fragments:
The genomic DNA is partially digested by an RE called Sau 3\/Mbo I (both REs have the same recognition sequences, i.e. they are isoschizomers).
This enzyme cuts the DNA every 256 base pairs on an average. The DNA is partially digested by this enzyme followed by gentle mechanical shearing. This generates average sized fragments, which can be cloned into 1 phage vectors.
The result is a number of random fragments with overlapping sequences. Cosmid and YAC vectors are also recommended for the genomic DNA library.
Joining, packaging and amplification:
Each DNA fragment is inserted into the 1 bacteriophage DNA to result in a recombinant DNA. Each recombinant DNA is packaged into a bacteriophage. This forms a primary library. The recombinant phages are plated on a lawn of E. coli grown in a culture. Infection’, replication and lysis of the host cells release a large number of recombinant
When the complete sequence of a gene is found in two different recombinant phages in the library, the screening is done in two steps: 1. the recombinant bacteriophages carrying the fragments of the gene are identified by colony hybridization and 2. the order of the fragments are determined by overlapping /common sequences between two fragments. This phenomenon has been termed as chromosome walking. 2. c (Complementary) DNA library:
Another approach for constructing a library is based on mRNAs of a cell. The mRNAs represent the structural genes of a cell at a point of time. All the cellular mRNAs are isolated and reverse transcribed to a single stranded c (complementary) DNA catalyzed by the enzyme, reverse transcriptase / RNA dependent DNA polymerase.
The cDNA is changed into a double strand by DNA polymerase enzyme. This double stranded DNA fragment is a copy of the gene that transcribed the mRNA. In this manner all the mRNA molecules are changed into double stranded DNA fragments.
This method enjoys several advantages over other methods. Eukaryotic mRNAs extracted from the cytoplasm are already processed, i.e. they are without non-coding sequences (introns), repetitive sequences and control regions. Secondly, the cytoplasmic mRNAs represented the expressed genes only and not all the genomic DNA.
Synthesis of cDNA:
There are two strands of the cDNA. The synthesis of the first strand is followed by the second strand.
Synthesis of the first strand: All eukaryotic mRNAs have a cap at the 5′ end and a poly-A tail at the 3′ end. The single stranded RNA forms the template for the synthesis of the first strand of cDNA. An oligo DT (a few deoxyribothymidylic acid residues) acts as a primer.
This olio DT fragment is base paired to the poly-A tail of the mRNA. Reverse transcriptase / RNA dependent DNA polymerase then carries out the elongation process by adding deoxyribonucleotides in 5′ -> 3′ direction. Deoxyribonucleoside triphosphates [d(ATP, TTP, GTP and CTP)] act as precursors in the elongation process. An mRNA: cDNA hybrid is formed.
cDNA hybrid is denatured to separate the mRNA. The 3′ end of the single stranded cDNA forms a hairpin that is used as a primer for the synthesis of the second strand by the Klenow fragment of E. coli DNA polymerase L Deoxyribonucleoside triphosphates are used as precursors. In the final step, the hairpin is digested by S, nuclease (DNA single strand specific nuclease). This results in a double stranded DNA.
II. Polymerase Chain Reaction (PCR) Based Cloning:
A specific fragment of genomic DNA can be amplified by a specific reaction known as polymerase chain reaction (PCR). PCR was discovered by Kary B. Mullis and F. Faloona in 1983. The reaction is carried out in a thermo cycler having a thermal cycling (heating and cooling) programme. The fragment of DNA, to be amplified, is put along with other substrates in the thermo cycler. After a recommended time period, the DNA fragment doubles at the end of the first cycle and quadruples after the second cycle and so on. In this cloning or amplification process, no host cell is used as the support system.
The reaction: The amplification of a segment of DNA takes place in the following three steps: (1) denaturation, (2) primer annealing and (3) extension. All the constituents of the PCR are put in a thermo cycler, programmed to an appropriate reaction condition. The input consists of the genomic DNA, primers (17 – 30 nucleotides in length), nucleotide precu1rsors and a DNA polymerase for extension.
Denaturation: The genomic DNA is heated at 94° C – 95° C for 0.5 min to render it single stranded.
Primer Annealing: The reaction mixture is cooled to 55° C for 1.5 min. Oigonucleotide primers (17-30 nucleotides) are annealed to the 3′ ends of both the denatured fragments. Primer selection is the most important part in the PCR elongation process. A suitable primer should have 50% GC. In some PCR elongation procedures, a degenerate primer is used. A degenerate primer is a mixture of primers, all having the similar sequence with variations at one or more positions.
Extension: The reaction mixture then is heated to 12° C for 1 min. The DNA polymerase elongates the new strand by adding nucleotides in a 5′ 3′ direction. Mullis and Faloona used the Klenow fragment of the DNA polymerase I in their original work. A fresh enzyme had to be used in each cycle of reaction because of the thermal denaturation step. Lawyer et al., 1990 discovered a DNA polymerase from the sulfur spring bacterium, Thermophilus aquaticus.