Amplification adjacent sequences

The basis of the RAPD technique is the differential amplification of genomic DNA. In the beginning of the reaction, the primer binds itself to the complementary DNA sequence, so the effective amplification takes place between two adjacent priming sites and a DNA polymerase enzyme promotes the extension if the orientation between them overlaps (see Fig. 12.8). 

The primers 5 -GGAATKn AAAGTTTTGTGTTATCTCA-3 and 5-TTAATATATGCdTATnTA-3 , employed for the amplification of human genomic samples, yields a 347 nt fragment from exon 4 and adjacent intronic sequences. Due to the small amount of genomic DNA sample available, these products were re-anplified by nested-PCR using the primer set 5 -biotin-... 

In order to sequence the inserted DNA by the dideoxy method , a suitable primer adjacent to the inserted DNA is required. One such primer, 96 nucleotides long, was obtained by introduction of a second EcoRl site in Ml3 mp2 to generate M13 mp2962. Annealing of this primer to DNA from a randomly selected M13 mp2 recombinant permits sequencing of approximately 200 bases to the 3 side of the 96-nucleotide primer. The method has been further refined by synthesis of a smaller (30 base pair) primer and its cloning into the plasmid vector pBR322 for amplification as required. With this primer, or the commercially available 12 base... 

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