i few days ago amplified specific gene using PCR with specific primers, but there was a multiple band in electrophoregram i found. out of 10 sample amplified, there was multiple band and there was a single band. i suspiciously observed this is an another gene. is that true or is this because something else ?
Please post a picture of the bands in the gel.
There can be many reasons for 2 bands....heteroduplex if amplifying a sequence with snps,poor primer annealing,existence of pseudogenes,genes in the same family as yours or that you are amplifying a common motif which is conserved in many genes. Good primer design usually is the answer to sorting this type of problem but often just increasing the annealing temperature of the primer annealing step of the pcr is sufficient. If your 1 and 2 bands are clean amplifications and the 2 bands are close together in size then a heteroduplex caused by a snp in the amplimer is possible
You should first check the specificity of your primers by running a BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). If your primers are really specific, you should try to optimize the PCR conditions (increasing the anneling temperature, modifying Mg++ concentration, primer concentration was too high, too much template was added or DNA contamination was introduced in primers or buffer).
Additionally, you should cut the expected band and sequence it to know if you are amplifying the gene of interest.
Tomi Kasayev In case is possible to make a gel extraction and try to sequence the no specific band?
Tomi Kasayev
- You could have also amplified isozyme genes (with different biological functions) or pseudo-genes or an area with 'repeats'. Pseudo-genes are nonfunctional segments of DNA that resemble functional genes [1] (attachment).
- It depends on how the band patterns and intensities look like on the gel. If there are just some 'light' bands scattering around in the gel lanes, then they are most likely the products of non-specific amplification (bad primers). As Paul Rutland mentioned, you should post your gel image.
[1] [ https://en.wikipedia.org/wiki/Pseudogene ]
Paul Rutland
By the way, I read this article (below) from Science two days ago. That reminded me of you doing genetic testing (PCR or sequencing) on very old museum specimens.
- The oldest genome (~1.2 million years old) has been sequenced. The DNA is preserved in the teeth of a Mammoth. Article Title: "Mammoth molars yield the oldest DNA ever sequenced [1]". I wonder how does the genome look like, compared to modern elephants...
[1] https://www.sciencemag.org/news/2021/02/mammoth-molars-yield-oldest-dna-ever-sequenced#:~:text=The%20oldest%20DNA%20sequenced%20from,lived%20some%20120%2C000%20years%20ago.
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