I have isolated full length cDNa of squalene synthase gene from root of tongkat ali. however I would like to validate my result whether the gene have intron or not. so I would like to isolate the genomic DNA of the gene and obtained the sequenced so that I can align with full length cDNA to see whether there is an intron or not.Is there any method that can be suggested?thank you
Hi there,
Southern blot derived techniques should be helpful using your cDNA as the probe. The easiest is to generate a genomic bank which you will probe with your cDNA to identify the positives clones.
The commonly used method is RACE-PCR. It allows to find up or downstream unknow sequences of a gene.
Design primers from 5' and 3'UTR of the cDNA and carryout genomic DNA PCR to amplify genomic fragment for the gene of interest. You may need to design nested primers from both the regions to be very sure that the fragment amplified is not a false fragment. Sequence amplified fragment and align sequence with cDNA sequence.
Hi,
You can prepare a genomic library of digested DNA and screen colonies with your prob. You can also prepare an enriched genomic library and clone only the size of gDNA that hybridize to your prob in southern blot. Cut out the region of interest and clone it regular plasmid like PUC19 . Then screen colonies with your clone. Alternatively you can use PCR with several primers on gDNA but for determining the right sequence, use proofreading polymerase and few cycles to avoid mistakes. If the gene has large introns primers on the 3' and 5' ends will not get results.
Good luck.
If your gene has no introns, then you should be able to use the same PCR primers that amplify the cDNA to amplify the corresponding fragment from genomic DNA. You can design primers for a series of overlapping short segments of the cDNA and use them on genomic DNA. If you find a location that will not amplify from genomic DNA or produces a longer fragment than expected, then this could mark the potential location of an intron. The attached pdf describing the SQS1 and 2 genes of arabidopsis shows you some other approaches too.
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