After cut single band from PCR-DGGE gel (534R, 341F-GC, which amplify a fragment of 193 bp ), then do PCR and ligation with pBlue vector, after transformation, I pick 10 clones and extracted the plasmid, and send the 10 plasmid sample for sequencing
Here are my questions:
1. The sequencing results are that these 10 clone samples have different sequence, does it mean the single band I cut from DGGE gel have at least 10 different type of bacteria?
2. When blast the product sequence online, some sample’s highest identity are below 90%, and some are 99%, I wonder how to determine the type of bacteria according to the identity?
3. It happened that some of the sequences had small size (150bp or 135bp) than the expected ( 193 bp), Could this be a sequencing error or possible contamination or some other reason? thanks!
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Xin
Principally, a single band in DGGE represents DNA from one species, the differences in similarity percent of the sequences may represent errors during PCR amplification, use high fidelity polymerases during amplification.
First some generalities about DGGE and pcr. DGGE works by using a GC clamp to bind one end of the pcr product so that the other end or melting domain can partially melt without the whole pcr product falling apart. This means that a change of base in the melting domain causes the whole pcr product to melt a little and to stop running through the gel compared with the normal sequence so that any band stopping in a different position from that of the normal sequence should have one,or more , changes in its melting domain. Unfortunately a number of different changes can all cause the band to melt and stop at the same position. The band stops in the gel at its melting position not at a position related to its size. It can be impossible sometimes to see if the altered ban has only one ,or possibly more changes in the melting domain because the first change stops the band moving further through the denaturing gradient.
The second point is the pct. Most pcr enzymes are best at 72c…this is the best temperature where the enzyme makes fewest errors. So the ideal pcr goes 94c then 72c only. In real life the primers anneal at low temperatures like 55c so the pcr goes 94c,55c,72c. Unfortunately the enzyme works at 55c but makes far more errors at the low temperature so the pcr process introduces many errors randomly ( but usually near the ends of the sequence ) every cycle. This usually does not matter for sequencing as the incidence of any change is low and the average sequence is the strongest signal.
The exception to this is when you clone the product then every clone only has one sequence so the pcr errors in the insert are faithfully replicated and sequenced. This makes it difficult to say if one sequence is a pcr error or a polymorphism of one common bacterial sequence or is a real change in a different species. Only sequencing a lot of clones will give a better idea of this. That is the advantage of sequencing pcr product directly as only the common changes show as polymorphisms and you never see the very low frequency pcr induced errors.
To identify if the changes are species related you could blast the sequences at the bacterial genome database at NCBI which has many thousands of genomes but obviously not all bacterial genomes
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