Technology

Advanced PCR Strategies

• Nested PCR. Many of the Company’s DNA analysis processes begin with a “nested” PCR step. We design a total of four oligonucleotde primers that will prime outside the target genomic region. In the first PCR step, the “outside” pair of primers is used to amplify the target sequence. Then, in a second, separate PCR procedure, the “inside” pair of primers is used to re-amplify the target sequence. The specificity of these two PCR steps is far greater than what can be achieved using only a single PCR step. This nested PCR strategy results in a higher PCR success rate, a higher purity amplicon product, and a more uniform product yield. It also allows us to specifically amplify targets that may be highly homologous to other regions of the genome.
  
  
• Long-Range PCR. For DNA haplotyping and mitochondrial analysis, the Company performs “long-range” PCR in order to produce amplicons of up to 10 kb in size. We use the Takara LA Taq DNA polymerase along with proprietary protocols for maximum PCR success. Large amplicons are then purified by gel electrophoresis. For haplotyping work, these amplicons are then cloned using Invitrogen’s TOPO XL PCR Cloning kit, which is designed for large insertions.
  

• ABI 3730XL. For Sanger DNA sequencing, we use fully automated, capillary-based ABI 3730XL DNA sequencers. This technology is the most thoroughly validated sequencing platform. Read lengths of up to 1000 bp are available. It has relatively short prep-times and run-times, permitting fast project turn-around times. It is the most cost-effective platform for relatively small projects.
  
  
• Roche 454 GS Junior. For larger amplicon sequencing projects and many special applications such as transcriptome sequencing, we recommend the Roche 454 DNA sequencing platform. This sequencing technology is more efficient for many larger projects because it can be multiplexed with respect to both samples and targets. Sample multiplexing is accomplished by introducing sample-specific tags by ligation or PCR. Each sample/target combination is sequenced multiple times, and a consensus sequence is determined for each combination. Each individual read preserves the haplotype phase of that amplicon, which is helpful for applications such as HLA typing. By using fewer combinations (and therefore higher coverage per combination) low-frequency variants can be seen in applications such as mutation analysis and mitochondrial sequencing.
  
  
• ABI 7900HT. For Taqman gentyping and for genomic sample QC, we use the ABI 7900HT Real Time PCR system
  
  
• Liquid handling and lab automation. Template amplification and DNA sequencing reaction setup are performed using high throughput liquid handling robotics. This allows thousands of samples to be analyzed quickly while minimizing human errors.