LA-PCRLong And Accurate Polymerase Chain Reaction
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Two methods DOP-PCR and LA-PCR have been used to amplify such a small amount of DNA from micro- dissected chromosomes.
For LA-PCR Sau3A1 specific linker with the sequences5'-GATCCGAAGCTTGGGGTCTCTGGCC-3' and 5'-GGCCAGAGACCCCAAGCTTCG-3' were used for construction of the Sau3A1 linker adaptor (Lowe et al.
To select the recombinant clone size larger than 300 bp obtained clones after DOP and LA-PCR were PCR amplified using M13- or linker-primers respectively.
PCR amplification of the micro-dissected chromosomes yielded products ranged from 100-2000 bp in DOP-PCR and 100-2500 bp in LA-PCR respectively with predominant fragments size from 300 to 2000 bp while positive control DNA was amplified between 100 to 3000 bp in both PCR methods.
Furthermore, we were isolated the [beta]-casein gene containing the mutant TATA box (CATAAAA) by LA-PCR of samples from another individual pig (Figure 2A).