Sequencing Primers

[jsmith613]

Gene sequencing involves the use of primers BUT how do we know which primers to use? How did the first ever scientists find out which primer was needed for the sequencing of the very first gene (when we knew NOTHING about any other gene)?

[canalon]

Clone in a plasmid/cosmid/BAC/YAC
Take a primer just beside cloning site (or better at both end)
Sequence
Design a primer at the end of your sequence(s)
Sequence
Repeat until you get the whole sequence, whether you go in 1 or 2 directions

This is known as sequence walking.

[DougB]

An early method used to determine an unknown DNA sequence was restriction mapping. Restriction enzymes are typically found in bacteria. These special enzymes will "cut" DNA into two pieces only where a specific DNA sequence is found. The DNA sequence could be 4 to 8 bases in length depending on the enzyme. Combining different enzymes provides a map showing where the specified sequences are located in an unknow sequence.

Restriction enzymes are also used to insert a piece of DNA into a plasmid, cosmid, BAC and YAC for cloning. Plasmids, etc., all have the advantage of containing restriction sites where the plasmid can be cut and an unknown piece of DNA is then inserted. Plasmid also have sites of known sequence where the initial primers are used to begin sequence and then primer walking to deteremine the entire unknown.

Hope this was helpful.

Doug

[JackBean]

This is very interesting question. The other one could ask, how did they determine the cut sequences?

[DougB]

Thanks Jackbean,

I guess that would be a good question. There are several possible methods for identifying the bases of an unknown region. One method is to synthesize a single stranded piece of DNA with a radioactive or fluorescent label as a probe. In ligation, the probe would only anneal to complimentary regions and identifies the presence of the known sequence.

Maxam-Gilbert sequencing provides another possibility. In this method, DNA is cleaved only at specific bases with chemical treatment. The resulting pieces are then run on gel to determine size. It is similar to restriction mapping. However, the bases do not need to be known. Dimethylsulfate is used to determine purines (G and A). Guanosine is affected more than Adenosine and gel bands appear darker on a gel. Hydrazine works for pyrimidines (C and T). Only Cystine bases are cleaved in high salt and are distinguished from Thymine bases.

DougB