1. Overlapping peaks occur when PCR products are sequenced
Problem Figure 1 (There are base deletions in the template, often single site (1-1) or two sites (1-2) base deletion leading to sequencing
Result shift code)
Solution: clone the PCR product into a plasmid (such as a T vector), select for monoclonal sequencing, or perform PCR on the PCR product.
Purification (at least agarose is fully electrophoresed and then gel-purified) and then sequenced.
Problem Figure 2 (PCR products are not pure, containing more than two fragments with partial sequences, varying in length)
Solution: The main reason is that the PCR product is not purified, and there are two or more fragments of different lengths with partial sequences, and the PCR products are subjected to PAGE.
Purification (at least after agarose is fully electrophoresed and then gel-purified) can be solved by sequencing.
Problem Figure 3 (Sequencing primers have base deletions)
The sequencing primer has a base deletion (generally a 5-terminal deletion of the primer), and the base deletion of the template is shown in Figure 1.
Some are similar. The difference is that the template base deletion usually occurs after a normal sequencing sequence. If the primer base is missing, the frameshift occurs from the beginning of the sequencing. The surface is graphically the beginning. Severe peak shapes overlap.
Solution: Re-synthesize the primers or PAGE purification of the primers
2. Peak overlap occurs when cloning and sequencing
Reason: The selected recombinant is not a monoclonal, and the plasmid for sequencing contains two or more different types of inserts.
Granules; or bacterial contamination for sequencing
Solution: Re-pick the colony of the monoclonal (single colony by single line), and then re-sequence the plasmid or the bacterial solution.
3. Samples have heterozygous/mutation sites
There is a heterozygous mutation in the template, which means that the template itself has a mutation at this site; or it is amplified from the genome.
Hybrid site. If the template is heterozygous (mutation or deletion), then the other sites in the sequencing pattern are generally single peak shapes.
Then suddenly there is an overlapping peak at a certain point (as indicated by the arrow in the figure).
Solution: It is recommended to clone the DNA fragment into the vector and then sequence.
4. Peak and sequencing signal attenuation caused by polyA/T and C/G cluster
RACE sequencing often encounters the situation in Figure 4-1 and Figure 4-4. Solution: Sequencing from the other end; but if such a sequence appears in the middle, there is currently no good solution, depending on the sequencing company's ability It is.
C/G cluster was encountered during sequencing of the PrV genome. Later, it was solved by TaKaRa. Although it didn't like devils, sometimes it had to use their products and services.
5, the gene contains repeats
Possible Cause: The sequencing results in the sample containing the repeat sequence are the same as the results of PolyA/T, resulting in Frame
Sliding, a shorter repeat sequence results in a frameshift in the sequencing result; a longer repeat sequence attenuates the sequencing signal.
Solution: Reverse sequencing can sometimes pass through the repeat region (but not necessarily), and the sequence results can be obtained by splicing multiple sequencing results.
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