The following guidance is provided for laboratories wishing to refer samples for DNA sequencing.

1. Host Strain

Different host strains yield varying quality of DNA template:

• DH5a & HB101 yield consistently good templates
• JM109/XL-1 Blue and MV1190 generally give good templates
• JM101 is generally a poor host

2. Primer

Between 5-20 pmoles of primer should be provided with each DNA sample to sequence. Lower concentrations will result in reduced signal. Higher concentrations may yield higher signals for some templates but will generally not provide much advantage for most situations. The primer should be from 17-30 nucleotides in length. There is no need for HPLC pure primers. The primer design is crucial and the TM should be between 50° C and 55° C. Other points of note:

• Using primers for sequencing that you have used in the original PCR can cause noisy initial sequence.
• G/C content should be between 40%-60%.
• Avoid stretches of any one particular base, especially G’s and C’s.
• Avoid runs of 3/4 G’s or C’s at 3' end. A high G/C content on a primer’s 3' end can result in unwanted priming events at other G/C rich areas on the template.
• A/T rich primers with low melting temperatures generally yield poor results in fluorescent DNA sequencing.

3. DNA

Plasmid

High efficiency of lysis is important (use fresh NaOH/SDS mix and freeze thaw culture prior to lysis) to avoid shearing of chromosomal DNA following lysis. It is important to remove contaminating RNAs - RNA background can cause random priming. ‘Quiagen’ P20 mini prep kits are recommended or possibly ‘Wizzard’ mini prep kits.

PCR Fragments

Efficient removal of amplimers is essential. Centricon 100 columns (Amicon) are recommended for this.

Quantity

Dilute DNA can give artifactual spectrophotometer readings. This problem is exacerbated if the DNA prep contains contaminating RNA and ribonucleotides. Running a gel with reference of known concentration provides a good estimation of DNA quantity and may help identify any potentially poor template (eg. salts, RNA contamination, chromosomal DNA contamination etc.)

Recommended amounts :

DNA

Single stranded 50-100 ng

Double stranded 250-500 ng

PCR 30-90 ng

Cosmid/l 2 m g

4. Sample requirement

The sample should be sent in a 0.5ml thin walled PCR tube. The appropriate amount of DNA and primer should be made up to a volume of 16m l with water (e.g. Milli-Q). Mineral oil should then be added to the tube (40m l).

Samples can be sent by post at room temperature to:

Mr Peter Martin

IBGRL,
NHSBT 
North Bristol Park,
Filton
Bristol
BS34 7QG

For further information contact Peter Martin:

Tel 0117-921 7572

Fax 0117-912 5782

e-mail pete.martin@nbs.nhs.uk