Same forward and reverse primer,s amplified DNA Product fails in Sanger sequencing, why it so.?
Ajay Saini
Hello Dr. Binoy,
In ISSR (and also in many multi-locus marker techniques like RAPD, AP-PCR etc.) one primer is used in the PCR amplifications for genotyping.
1) In such cases the PCR will work but the same primer when used for sequencing will extend both the templates ...as a consequence the reaction will yield mix sequence.
2) The ISSR fragments have SSRs at ends and may of these sequence motifs are not good to serve as primers, they may also bind to overlapping regions in the template
So it is better to clone the desired ISSR fragments and use vector specific universal primers for sequencing
Ajay
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Laurence Stuart Dawkins-Hall
- Agree with Ajay
- I would also add that unless you column/gel purify your PCR products sufficiently, you will get carryover of residual dNTPs, primers and/or residual salt:
- Residual dNTPs will cause your sequencing reaction trace to be 5' top heavy and shorten your read; residual primer (especially if both PCR primers) will cause a double sequencing ladder, rendering the chromatogram unreadable and salts will inhibit ampli taq, resulting in an un readable trace owing to poor signal to noise
- To distinguish between a poor trace due to primer contamination and one due to poor signal to noise examine the raw data: In the 'contaminated' trace it will be strong whereas a trace where cycle sequencing reaction inhibition has occurred the raw data will be weak
- As an alternative to gel extraction or column purification (to remove dNTPs primers and salt) you can digest your PCR product with shrimp alkaline phosphatase and Exonuclease III (SAP exo): SAP deactivates dNTPs and exonuclease degrades your primer; Find attached an SOP
- As mentioned Ajay another potential problem in terms of unreadable sequence is the nature of the target sequence; Namely repeat motifs: These can cause ampliTaq to stall detach and therefore reduce the efficiency of your cycle sequencing reaction and hence the strength of the resulting sequence trace. Alternatively, repeat sequences rich in A/T bases can cause production of multiple out of register sequencing ladders which over lap and render the sequence unreadable. This is caused slippage: To reduce this phenomenon you can prime and extend at a lower temp (cf. 55C-60C) instead of priming at this temp and extension at 72C. See attached also
- https://www.researchgate.net/post/How_to_sequence_a_10kb_gene_with_repeat_regions#view=5893803eed99e14186574c3a
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