I subjected some genomic DNAs to PCR reaction and later sent then for sequencing to analysis for polymorphisms in my gene of interest? Some of my sequence results came out fine while some came out analyzable due to noise or mix. Any suggestion on what could be responsible for this and what could be done to resolve this?
In my point of view the possible reasons are:
(1) Dirty PCR amplification, means two pcr sequences are present ...check the pcr product on gel for single product
(2) Presence of traces of primers present on sequencing reaction (Proper purification required before sequencing)
(3) Old or dirty capillaries of sequencer
I agree with the three points given by Sandeep Kumar. Be sure to clean up the PCR product, for instance using ExoSAP-IT.
In addition, this site may give some useful answers.
http://ampliconexpress.com/troubleshooting-dna-sequencing-evaluating-sanger-dna-sequencing-chromatogram-data/
- I think the problem probably isnt contaminating sequence as your are amplifying from genomic DNA so unless your primer is biding to a secondary region - say a pseudogene - that is less likely
- What is more likely to give noise in your sequencing ladder is low signal to noise due to low raw signal from sequencing ladder
- This can be a function of certain primers regardless of whether they are made well or not so my advice would simply be to design another set of primers and sequence those regions again
- If the noise due to poor signal amplitude is sample specific then subject your sampple o another genomic DNA extration os clean up your existing gDNA sample with Trizol chloroform follwed by ethanol precipitation or simply ethanol precipitate your sample and subject to 2 x 70% ethanol washes before re sequencing: This is because salt contamination in genomic DNA can inhibit the AmpliTaq in big dye sequencing reactions so removing that with 70% ethanol migh improve your sequence quality from those samples
- To improve the quality of Big Dye sequencing reactions in general (by increasing signal to noise):
can you attach an original .ABI sequence file for one good and one poor sequence please?
What pcr product purification system are you using prior to sequencing or is a company doing this step for you?
Are all of the poor sequences from one primer and the good ones from another or are they all the same orientation (forward or reverse) sequences?
How does the raw data look like? Lower or higher signal when comparing "good" and "bad" sequences?
Did you measure amplicon concentrations before sending them out to be sequenced? Checked in agarosegel? Did all amplicons have the same brightness?
Hi
Probably, there is a common indel with high frequency in your PCR product. Check the sequence and find where the noise got started, probably there is a polymorphic indel (insertion/deletion) in that position. You can find this indel in SNP database. Go to SNP database in ncbi then type your gene name or genomic position then limit SNPs to indels for your PCR product regions.
Good luck
1 - One of the main causes of this event is the DNA contamination after/during the extraction and not less important the contamination of the product during DNA purification after PCR amplification, to be sure you don't have a product contaminated you do a quality control through electrophoresis and not only verify quantitavely by spectrophotomether.
2- Another reason is to check the quality of the primers you are using and take care not utilize EDTA (included in some sequencing kit) because affect the dna pol by taking out the Mg2+ from the polimerase and can affect the sequencing.
3- To prevent the sequencing noise is avoiding contaminants during DNA extraction and purification after PCR and doing sequencing of reverse DNA chain to notice if that noise region in the forward chain is really a noise and always taking account the standart sequencing downloaded from gene databases.
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