We are planning to detect the number of GT repeats in the promotor of a specific gene. Our specific aim is to correlate the number of repeats with protein levels measured previously wit ELISA. We amplified the target sequences using selected primers in which the sense primer is labelled with a 5-carboxyfluorescein. However, the results obtained from sanger sequencing are completely different sequences. I blast the resulted sequences and no much with my gene has been found. Please anyone has an experience in another method for repeat detection is highly appreciated to comment.
You can differentially separate small DNA fragements with single base pair differences on thin PAGE gels....think ABI 377 sequencer. Design primers immediately adjacent to the repeat and amplify with a hi fidelity polymerase. With the short length of the fragment you should be able to reduce your PCR extention time to favour amplification of the shorter target sequence over non-specifc sequences. You could even generate your own ladder using primer synethsis to generate the amplicon with various lengths of GT repeats??.....just an idea!!
If gene and promoter sequence is known than just counting by eye or by script
alternatively
Sequencing this region.
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