1.The importance of DNA cloning:
Current DNA technology is based on two different approaches:
a. Specific amplification (DNA cloning) which involves cell-based DNA cloning (involving a vector/replicon and a suitable host cell) and in vitro DNA cloning (PCR)
b. Molecular hybridization where the DNA fragment of interest is specifically detected using a mixture of different sequences
2. Polymerase Chain Reaction (PCR): features & Applications:
Template DNA: DNA (linear or circular) or cDNA (complementary DNA produced from produced mRNA by reverse transcriptase)
Primers: pairs of oligonucleotides each 18-25 nucleotides long; 40%-60% GC content; melting temp of both should not differ by >5oC; 3’ terminal sequences of any primer should not be to any sequences of the other primer in the pair; self-complimentary sequences (inverted repeats) of
>3 bp should be avoided.
Cycling nature & exponential amplification: denaturation; primer annealing; and DNA synthesis (extension).
Regular Taq DNA polymerase lacks 3’ -> 5’ exonuclease activity needed to provide proof-reading function.
