Various biological tools essential in rDNA technology include:
i. Instruments: PCR, Agarose Gel Electrophoresis, SDS-PAGE
Polymerase Chain Reaction (PCR) is a vital process for amplifying the gene of interest in vitro using a PCR machine. It can generate billions of copies of a specific DNA or RNA segment with high accuracy and specificity within a few hours. The PCR process is automated, involving thermal cycles for denaturation and renaturation of double-stranded DNA. The necessary device for PCR is a thermal cycler.
Requirements for polymerase chain reaction include DNA with the desired segment, deoxyribonucleoside triphosphates (dNTPs), primer molecules, heat-stable DNA polymerase, and appropriate quantities of Mg++ ions.
ii. Biological tools: Enzymes, Cloning Vectors, Competent host
Different enzymes play crucial roles in rDNA technology, including lysozymes, nucleases (exonucleases, endonucleases, restriction endonucleases), DNA ligases, DNA polymerases, alkaline phosphatases, and reverse transcriptase.
Enzymes like exonucleases and endonucleases cut nucleotides from DNA ends or within the DNA strands. Restriction endonucleases, or restriction enzymes, act as molecular scissors by cutting DNA at specific sequences known as recognition sites.
Cloning vectors in rDNA technology must possess certain characteristics, such as the ability for independent replication, easy introduction into host cells, marker genes for antibiotic resistance, a unique cleavage site for a restriction enzyme, and suitable control elements like promoters and ribosomal binding sites.
Examples of constructed plasmids used as vectors include pBR322, pBR320, and pACYC177.
Competent hosts, often bacteria like Bacillus haemophilus, Helicobacter pylori, and E. coli, are used for the transformation with recombinant DNA in rDNA technology. E. coli is a common choice for this purpose.