OHSU

Sequencing Notes

Linear Amplification using Sanger Cycle Sequencing

This technique uses a thermostable DNA polymerase, buffer (with Mg+2), dNTPs and dideoxyNTPs to generate a family of extension products that can be separated by electrophoresis and used to "read" the DNA sequence of the original template. 

For the history and development of the technique, check theses links from the NCBI Bookshelf.

A MolBio 101 description can be found here.


SNP Discovery and Confirmation

SNP

Single nucleotide polymorphisms are the most abundant form of genomic sequence variation (~1 every 1000 nts in H. sapiens). SNPs are thought to be important for understanding variation within populations.

We offer free sequence comparison using the University of Washington suite of sequence analysis tools, including PolyPhred for SNP analysis.

 


Epigenetic Sequence Analysis

Epigenetics is the study of heritable changes in gene expression not due to alteration of the DNA sequence. It involves DNA methylation and histone protein modifications. The most common epigenetic modification is the methylation of C nucleotides in CpG sequences in gene promoter regions. Methylation is a normal cell process, the enzyme involved is DNA Methyl-Transferase which converts "regular" Cs to 5-methyl-Cs. 

The reagent Sodium Bisulfite can be used to convert unmethylated Cs to Us, which are replaced by Ts during replication (cloning) or amplification (PCR). 

C_conversion_U

Sequence comparison of treated vs untreated DNA is used to discover epigenetic C modifications.