9 replies to this topic

[proteinz]

hi all!
has any one ever seen two only slightly different gDNA sequences ( sizes only 3bp in difference) running as two distinct bands on an agarose gel before? i have attached the picture... why would this occur? both bands were sequenced and vary only slightly in sequence in the intron region.
thanks in advance!

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[eldon]

proteinz, on Aug 2 2009, 05:46 AM, said:

hi all!
has any one ever seen two only slightly different gDNA sequences ( sizes only 3bp in difference) running as two distinct bands on an agarose gel before? i have attached the picture... why would this occur? both bands were sequenced and vary only slightly in sequence in the intron region.
thanks in advance!

not sure what you mean. they look to be different by ~ 100bp.

need more info.

is it duplex pcr? two primer pairs?

[HomeBrew]

Is this really genomic DNA ("gDNA"), at that size?

[dreww]

i doubt it

[little mouse]

There is more than 3 bp of difference. Could be that the difference of length is just before the primer you used for sequencing, that's why you don't see a difference by sequencing.

[proteinz]

cheers for the replies guys!
i used a primer pair from the ORF region i.e. the start to stop codon of my gene to amplify gDNA (to identify any intron exons within the gene). i got two bands from the resulting PCR when i cut them out, purifed and got the bands sequenced it arrived back with the primers present at the start and end of the gene with only a 3bp size difference

[Tintin]

proteinz, on Aug 3 2009, 08:28 AM, said:

cheers for the replies guys!
i used a primer pair from the ORF region i.e. the start to stop codon of my gene to amplify gDNA (to identify any intron exons within the gene). i got two bands from the resulting PCR when i cut them out, purifed and got the bands sequenced it arrived back with the primers present at the start and end of the gene with only a 3bp size difference

Fully sequence or only 5' and 3'? Try sequence it with both primers and make sure you have the whole sequence. And sometimes sequence company mess up your sample (sucks but it did happened to me before), did they sequence one sample twice?

[proteinz]

i see where your coming from tintin but i dont think they did it twice coz i sequenced each band forward and reverse 3 seperate times and then annotated the samples together using CAP3 program. they are not fully identical they contain variable intron regions. any other ideas?

[eldon]

proteinz, on Aug 3 2009, 07:55 AM, said:

i see where your coming from tintin but i dont think they did it twice coz i sequenced each band forward and reverse 3 seperate times and then annotated the samples together using CAP3 program. they are not fully identical they contain variable intron regions. any other ideas?

there is no way that those 2 bands differ by only 3 bp. you cannot resolve 3 bp on an agarose gel (what is the % of that gel? 1%?)...you would need to use polyacrylamide.

you need to sequence from both ends and you might also need to sequence the larger fragment using different primers ~ 300 bp downstream of the first pair of sequencing primers to ensure the accuracy of the reads.

it is possible that you or your sequencing center sequenced the same fragment hence 3 bp difference...and again you need to thoroughly check the electropherograms to ensure the bp calls that differ btwn the two sequences are accurate.

otherwise, you might have 2 copies of that gene that differ by ~ 100bp (e.g., an extra intron or exon).

Edited by eldon, 03 August 2009 - 08:42 AM.

[proteinz]

thank you for your kind help guys...il go back to the drawing board!