ABRF '96: Biomolecular Techniques

Tutorial #4

SEQUENCING DIFFICULT TEMPLATES
Margaret Robertson
University of Utah

Monday, April 1, 1996
10:50 AM -12:20 PM
California Room
Holiday Inn Golden Gateway
San Francisco, CA



Contents:
  1. Factors that affect the quality of sequencing.
  2. Types of difficult templates.
  3. Sequencing PCR products.

1. Factors that affect the quality of sequencing.
Purity of DNA - use robust template preparations. Recommended in order of reliability:
Qiagen tip 20, tip100
Alkali lysis/PEG precipitaion
CsCl banding
Wizard, 5' 3', SNAP columns.
Classical alkali lysis
Host strains
DH5alpha and HB101 work well
Common contaminants
EDTA at concentrations > 0.5mM - compromises Mg concentration.
Phenol - quenches fluorescent dyes.
High salts - inhibits enzyme.
SOLUTIONS:
DNA QUANTITY
Taq FS requires less template and seems to tolerate a larger window of template concentration.


ssDNA
dsDNA
PCR<1Kb
PCR>1KB
Cosmids, P1 clones
DYE P
100-300ng
300-600ng
6-60ng
50-150ng
n/a
DYE T
100-300ng
150-300ng
30-60ng
50-150ng
1-2ug

5ng/100 bases has been suggested by some as general guidelines for PCR products.

PRIMER QUALITY

Primers should have most of the following characteristics to be able to produce good, consistent sequencing data.

2. TYPES OF DIFFICULT TEMPLATES.

Be prepared to use all possible sequencing chemistries.
No SINGLE chemistry will work on all templates, although Taq FS does a very good job on most difficult templates.

High G/C Content

Conclusions:

Sequencing for heterozygote mutations.

This is probably one of the more demanding DNA sequencing procedures!