Question

EcoRI Hindi EcoV BamHI Son Sa Put Pasti Xmail -Nui 3 4. Describe how a recombinant DNA could be constructed using this plasmid and a PCR product that has been amplified with primers that have BamHl restriction sites on the 5' and 3'-ends. How would you identify cells that carry a recombinant DNA molecule? Be specific include all of the necessary steps. Write your answer in complete sentences

Answer 1

A recombinant DNA is defined as the hybrid DNA molecule produced by the combination of DNA from two different species to create a new genetic combination that is of value to science, medicine, agriculture, and/or industry.

The PCR (Polymerase Chain Reaction) is a technique that allows the amplification of a specific DNA region in vitro.

Plasmids can be defined as the small circles of DNA which are found in the bacterial species and also in some other organisms. These plasmids are capable of self-replicating, i..e, capable of replicating independently of the chromosome of the host cell.

The given plasmid in question has antibiotic-resistance genes, which are symbolized by Amp' and Tet', where Amp' means ampicillin resistance and Tet' means tetracycline resistance. This means that the plasmid can survive in an environment containing antibiotics like ampicillin and tetracycline. These antibiotic resistance genes can be used as a selectable marker to ensure that the bacteria which is produced after the recombination contains a particular plasmid.

In the process of synthesizing a recombinant DNA (rDNA), two types of enzymes are required - a restriction endonuclease and a ligase. The restriction endonuclease is required for cutting and the ligase is required for joining the nucleic acid molecules together.

The restriction endonucleases are enzymes that can cleave the double-stranded DNA at a limited number of specific recognition sites.

Thus, for the synthesis of the rDNA, the DNA must be cut in a precise manner which is also reproducible. Thus, the vector (i.e., the plasmid) is cut once at a single position to open up the circle so that the DNA fragment can be inserted. These restriction endonucleases have a specific recognition sequence or site where it can cut the DNA molecule. In the given question, it can be seen that the vector (i.e., the plasmid) has restriction sites for BamHI.

BamHI is produced by the organisms Bacillus amyloliquefaciens and the recognition sequence for this endonuclease is GGATCC.

Since the PCR product also has BamHI restriction sites at the 3' and 5' ends, the PCR product will also be cleaved by the restriction endonuclease. BamHI has the capability of producing sticky ends. Sticky ends are produced when the two strands of DNA are not cut in the same position. Thus, overhangs or short single-stranded regions are produced at each end. These sticky ends are cohesive and allow for the base pairing between the ends.

Thus, the pairing between the sticky ends of the PCR product and the sticky ends of the vector cleaved with BamHI is possible. Sticky ends increase the efficiency of ligation because these sticky ends can base pair with each other by hydrogen bonding. This forms a structure that is stable and thus allows the enzyme to perform better.

After this process of cleaving, the final step is the joining together of the vector and the PCR product (i.e., the DNA). This is the ligation process which is mediated by the enzyme DNA ligase.

After this rDNA has been produced by the above-stated steps, this rDNA molecule has to be inserted into the living cell which is capable of growing and dividing to produce the clones of this rDNA. The organism which is generally used is bacteria.

The process of transformation is where the rDNA (or generally, any DNA) is taken up by the bacterial cells. Generally, transformation is mediated by some type of chemical or physical treatment to the bacteria cells to increase their capability of taking up the DNA. The cells which have taken up the DNA are said to be competent cells.

Now, to detect whether the cells have effectively taken up the rDNA molecule or not, a selection procedure is carried out. The plasmid used as the vector contained genes for resistance to the antibiotic ampicillin (Amp') and tetracycline (tet'). If we consider that the bacterial cell E.coli has been used for the expression of the rDNA, then it is a known fact that E.coli is sensitive to the antibiotics like ampicillin and tetracycline. However, in cells that are transformed effectively, the presence of the antibiotic resistance genes of the plasmid will allow them to grow in a medium containing the antibiotics ampicillin and tetracycline. Similarly, for the bacterial cells which have not taken up the rDNA and are not transformed (i..e, the non-transformants) they will die when grown in a medium containing the two antibiotics. Thus, the transformants will grow and form colonies in the medium containing the antibiotics ampicillin and tetracycline, while the non-transformants will not produce colonies.

Thus, in this way, the effective selection of the transformed cells can be done.

To determine which of the transformed cells contain the rDNA, identification of the recombinants is important. In the given question, BamHI is the restriction endonuclease. BamHI cuts the vector (assuming pBR322) at only one position. This position is in the gene that codes for tetracycline resistance. Thus, the recombinant DNA contains the PCR amplified DNA in the BamHI site. So when the host which carries this rDNA is grown in a medium with tetracycline, it does not grow because the resistance to tetracycline is lost.

Thes cells will still show resistance to ampicillin but will lose the resistance to tetracycline.

In this way, the screening can be performed and the recombinants can be successfully selected by the method known as 'insertional inactivation'.