Hi, I wish to sequence entire gene of Streptococcus pneumoniae. The length is 2.2 kb. I wish to detect presence of any mutations or variations in this gen. Can I design a single set of primers flanking this region and sequence the amplified product by Sanger sequencing? What can be the maximum amplicon size for Sanger sequencing? Or should I design a few sets of primers within the gene also and amplify them?
We commonly sequence up to 800 bp for each primer with Sanger, and consider that the first 30-40 bp could be not very good (looking at the phred score in the outpput of seqencer, you can see it below the threshold of 20). In your case I suggest to start from -100bp from the start codon for the first primer forward (Fw1) and design a total of 4 primers (FW1-Rv1 - extreme - and Fw2-Rv2 - internal) that overlap themselves in the extremity (3' and 5' in the central part of the gene) to cover the entire gene. You can also amplify by PCR using only the external primers (Fw1-Rv1 to obtaining the entire sequence) and then use each primer for each Sanger sequencing and as template the PCR product (the entire gene). This will lead to have good quality sequence to investigate any SNPs and INDELs present in your sample.
Regards.
if you can amplify the entire 2kb in one pcr that is fine. Then design additional sequencing primers within the sequence at about 600base separation and use these to sequence off the 2kb template. the sequence will be unreadable in the primer sequence and for the next 30 bases so overlap the good sequence 600-700 bases with the new primers. The best you are likely to get with sanger sequencing is about 800 bases from each primer and the quality of sequence gets worse towards the end of the sequence ( low signal amplitude) so you want a good amount of overlap
Multiple primer sets might be used. The primers should yields 600bp length amplicon. Then after sequencing different sets of primers, original gene will be identified through overlapping method.
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