Question

I need help with both parts a and b of isolation and amplification. I would like both parts answered since it only counts as one question. Thank you.

Gene is SCN5A Gene

1. Isolation and amplification of an exon (>100 bp) of a gene by PCR (5 points)
   a) Isolate human genomic DNA
   b) Design PCR primers to amplify the exon and add restriction sites to both 5’ and 3’ of the DNA

Answer 1

a. For amplification of a gene , first step is isolation of the genomic DNA (human). There are various methods available for isolation of DNA e.g. phenol chloroform method, sodium perchlorate method etc. These are manual methods which need to be standardized in lab before initiating genomic DNA isolation for precious samples. In addition to manual methods some standardized methods are there for which various companies provide kits to do so. After isolation of DNA, the quality and quantity of DNA should be analyzed before starting PCR by gel electrophoresis and spectrophotometer respectively.    

b. The SCN5A gene has 28 Exons which span 80 kB of genome. Primers will be specific for each exon. The exons are separated from each other by introns in between them. So, primers specific for each exon will amplify that particular exon exclusively. The exons are identified as exon 1, exon 2, exon 3 and so on... So, we need to identify that which exon we need to amplify depending o the need of experiment. Primers can be designed for each exon exclusively via various tools like NCBI primers, UCSC genome browser etc.