4 replies to this topic

[jiajia1987]

Dear peeps,

I am doing an error-prone PCR and I am interested in introducing mutations in my gene. I have two pairs of primers flanking my gene, which is nearing 2kb in length. Does anyone know on average, Taq introduces a wrong nucleotide during extension after approximately how many base pairs? I have been trying to find this in journals, but I am unable to find any. please help!!!! !!

Thanks in advance!!

[mastermi]

I think it's one in a thousand. If you want to produce a lot of point mutations better use additional mutational conditions.

[jiajia1987]

mastermi, on Apr 18 2009, 01:12 AM, said:

I think it's one in a thousand. If you want to produce a lot of point mutations better use additional mutational conditions.

Thanks for getting back to me. I am sorry for this late reply.

I am using MnCl2 and biased dNTPs to increase the mutations

[HomeBrew]

jiajia1987, on Apr 17 2009, 10:25 AM, said:

Does anyone know on average, Taq introduces a wrong nucleotide during extension after approximately how many base pairs?

This is known in other circles as the "gambler's fallacy". If the rate of misincorporation is 1 in 1000, then *every* base has a 1 in 1000 chance of being wrong, just as there's no better chance of getting a good card hand just because you lost the last six hands in a row.

This assumes Taq is not more likely to misincorporate one base over another.

There are some error rate tables here, but reported error rates vary because they are condition dependent.

[jiajia1987]

HomeBrew, on Apr 21 2009, 10:30 PM, said:

jiajia1987, on Apr 17 2009, 10:25 AM, said:

Does anyone know on average, Taq introduces a wrong nucleotide during extension after approximately how many base pairs?

This is known in other circles as the "gambler's fallacy". If the rate of misincorporation is 1 in 1000, then *every* base has a 1 in 1000 chance of being wrong, just as there's no better chance of getting a good card hand just because you lost the last six hands in a row.

This assumes Taq is not more likely to misincorporate one base over another.

There are some error rate tables here, but reported error rates vary because they are condition dependent.

Thanks for the error rate table.