454 pyrosequencing amplicon variant analysis software  (Pyrosequencing Inc)

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: Primer design

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Software, Sequencing, Amplification

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: Primer design. As one can see in the Design Template, there is a padding region of 300 base pairs (bp) before and after the exon where the forward and reverse primers can be designed. The amplicon includes the target region which in turn consists of the region of interest (exon and a padding of 15bp important in the splicing mechanism) and a region of 5-35 bp that is technical requirement of the 454 or Sanger respectively to give accurate sequencing results. Each primer (P1 and 2) contains a specific and a universal region (black and red regions). The specific region attaches to the gene of interest. This primer region is used during the amplification process (PCR) and becomes part of the amplicon. The universal primer used during the sequencing process hybridizes to the universal region allowing the sequencing of any amplicon.

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Amplification, Sequencing

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software. The different intensity of the superposed bases in the Sanger chromatograms of the different patients illustrates the difficulty for an automatic call with SNP detector. On the other hand, in the 454 pyrosequencing, the mutations are successfully called by AVA. For this reason, the 454 pyrosequencing is a great discovery technology. However, the % given is not representative of the % of patients with the mutations because of factors such as heterogenous sample, heteroploidy and chromosomal loss. Genotyping with another technology then needs to be performed in each sample as well as in the matched normal tissue to determine which mutation is somatic and define the percentage of patients with each mutation.

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Amplification, Variant Assay, Software, Mutagenesis