Aiding DNA Amplification of GC-rich Regions in the Human Genome for Illumina Sequencing
Mentors: Dan Aird and Andi Gnirke
Next-generation DNA sequencing technologies such as the Illumina Genome Analyzer allow cost-effective sequencing and re-sequencing of whole genomes including the human genome. However, regions that are extremely GC-rich (i.e. contain many more Gs & Cs than As & Ts) are often under-represented or completely absent in “whole-genome” sequencing data. Polymerase chain reaction (PCR) is a critical step in the DNA sequencing process, but PCR amplifies GC-rich regions of DNA poorly. Jeff and his mentors aimed to find new PCR conditions that amplify every part of the genome evenly.
Through this work, Jeff discovered five combinations of PCR conditions that worked well to amplify GC-rich DNA. His best condition, using a non-standard polymerase enzyme with the additive betaine and an 80-second long denaturation time, worked well with both long and short pieces of DNA. This specific combination showed a 50-75 fold improvement over standard conditions, and thus will be utilized in the DNA sequencing process to see if it can be used to sequence all regions of DNA in a genome equally.
Jeff, a senior at Cambridge Rindge and Latin School, optimized conditions for sequencing DNA from GC rich regions of the genome.