OHSU

DNA Sequencing User's Guide

Overview of Automated DNA Sequencing

During sample preparation, the DNA fragments in a sample are chemically labeled with fluorescent dyes. Using PCR, unlabeled deoxynucleotides (dNTPs) and dye-labeled dideoxynucleotides (ddATP, ddGTP, ddCTP, and ddTTP) are incorporated by Taq DNA polymerase into the growing DNA strands. The dye-labeled DNA fragments are separated by electrophoresis within the capillary array of the ABI Prism 3130xl Genetic Analyzer. Once the fragments enter the detection cell, they pass through a laser beam. The light excites the attached dye labels causing them to fluoresce. A computer analyzes the fluorescence to determine the order of DNA bases in the strand. Two files are created for each sequence: a text file with the extension *.seq and an electropherogram (a graph of relative dye concentration against time, plotted for each dye) with the extension *.ab1.

 

SAMPLE PREPARATION

Culture

  • ABI recommends HB101 or DH5a.
  • Mv1190 and XL1Blue give variable results.
  • JM101 usually doesn’t work.

Purification

Recommended for use: Use a Qiagen kit or standard alkaline lysis miniprep purification followed by 13% PEG8000 precipitation. Resuspend DNA in dH2O.

What to avoid: Do not use Promega Magic preps. Avoid purifying DNA with phenol since residual phenol interferes with the sequencing reaction. Do not resuspend DNA in TE.

In general, whatever purification process is easiest to prepare should be fine. However, the cleaner the template provided, the cleaner and better the sequence read and read length returned. You are responsible for the purity of your samples...do NOT send contaminated samples!

Primers

Longer primers work better. T7, T3, and M13 forward and reverse primers are recommended. SP6 is less reliable. Custom primers should be 21- or 22-mers.

Preparing Samples for PCR

Recipe for the CLEAR pre-mix (or Dye Terminator Mix) reaction

Reagent   Quantity
Template ss DNA
ds DNA
0.05–0.10 μg
~0.50 μg
PCR product 100–200 bp
200–500 bp
500–1000 bp
1000–2000 bp
>2000 bp
1–3 ng
3–10 ng
5–20 ng
10–40 ng
40–100 ng
Primer
3.2–6.4 pmol
dH2O
quantity sufficient
Final volume
9 μl

Mixing Instructions:
Mix reagents in a small, thin-walled tube labeled with your initials and a unique number (e.g., PIB1). Label each sample horizontally above the line of the tube—do not include your primer or template on the label. Writing anywhere else, including the lid, will be rubbed off in the PCR machine, forcing you to repeat your sample. The Core will add Dye Terminator Mix before running your samples.

NOTE: For Double-Stranded Plasmid DNA, increasing the amount of primer more than 6.4 pmol generally doesn’t cause problems. But don't add too much! For PCR products, however, it is generally better to stay around 3.2 pmol or only slightly higher if the PCR product is very large.

 

ANALYZING DATA

Viewing Sequencing Text File

Open the appropriate *.seq file using TextEdit on a Mac (OSX) or Notepad on a PC. A dialog box will open and the sequence can be copied & pasted into Intelligenetics, GCG Strider or other software.

NOTE: if there is no *.seq file and the electropherogram (*.ab1 file) is 72K, your sample failed to give a readable sequence.

Analyzing Electropherograms

Several free programs are available through the Internet to view the *.ab1 file: