To whom working with DNA sequencing - (Feb/29/2008 )
1. Would a primer extension reaction in DNA sequencing work using an RNA polymerase? any one can explain this point please?
2. When the products of a DNA sequencing reaction are separated by electrophoresis, why is it normally only possible to read about 500 to 800 bases, yet the sequencing reaction continues for thousands of bases?
-roniadam-
QUOTE (roniadam @ Mar 1 2008, 07:36 AM)
1. Would a primer extension reaction in DNA sequencing work using an RNA polymerase? any one can explain this point please?
On first glance, no. RNA polymerase doesn't extend primers made of DNA. Nor will it use the dNTPs much less modified ddNTPs. On second glance...
Google search indicates RNA dependent RNA polymerase, from RNA viruses will extend RNA primer Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase. Not quite what is desired.
This paper ,Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction indicates mutant RNA polymerase will extend a DNA primer... but only upto 8bp.... so potentially such mutant might accept ddNTPs but it will probably go not futher then 8bp probably less.
QUOTE (roniadam @ Mar 1 2008, 07:36 AM)
2. When the products of a DNA sequencing reaction are separated by electrophoresis, why is it normally only possible to read about 500 to 800 bases, yet the sequencing reaction continues for thousands of bases?
Skill and good equipment. The skill is the bit of black arts of get the reaction to produce just the right quantities of product. The equipment... I can't say. I have never worked the machine before.
When I started I could only get my useful sequencing reads to 500bp, with practise I got it up to 800-900bp. However a labmate of mine (a post doct) can get a useful sequence reads upto 1200bp regularly. I seem to recall a person here reporting he obtained a read upto 1400bp.
Nevertheless, the sequencing reads probably can't go on for thousands of bp Since a DNA stand can be terminated with each additional bp, the probability of a DNA strands being sythesised decreases with its lenght. Thus the quantity of a particular DNA stand present decreases with its lenght. This in turn puts a cap on how long a read can go. Once the quantity of DNA synthesis become so low the machine is no longer sensitive enough to detect the signal. I think there is also the problem of separating DNA product that differ by 1 bp... the spacial separation between DNA band becomes less as the band size increases. Setting an upper limit of band resolution (at a particular gel density). And therefore identity of the next bp in a long DNA stand.
-perneseblue-
QUOTE (perneseblue @ Mar 1 2008, 07:07 PM)
QUOTE (roniadam @ Mar 1 2008, 07:36 AM)
1. Would a primer extension reaction in DNA sequencing work using an RNA polymerase? any one can explain this point please?
On first glance, no. RNA polymerase doesn't extend primers made of DNA. Nor will it use the dNTPs much less modified ddNTPs. On second glance...
Google search indicates RNA dependent RNA polymerase, from RNA viruses will extend RNA primer Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase. Not quite what is desired.
This paper ,Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction indicates mutant RNA polymerase will extend a DNA primer... but only upto 8bp.... so potentially such mutant might accept ddNTPs but it will probably go not futher then 8bp probably less.
QUOTE (roniadam @ Mar 1 2008, 07:36 AM)
2. When the products of a DNA sequencing reaction are separated by electrophoresis, why is it normally only possible to read about 500 to 800 bases, yet the sequencing reaction continues for thousands of bases?
Skill and good equipment. The skill is the bit of black arts of get the reaction to produce just the right quantities of product. The equipment... I can't say. I have never worked the machine before.
When I started I could only get my useful sequencing reads to 500bp, with practise I got it up to 800-900bp. However a labmate of mine (a post doct) can get a useful sequence reads upto 1200bp regularly. I seem to recall a person here reporting he obtained a read upto 1400bp.
Nevertheless, the sequencing reads probably can't go on for thousands of bp Since a DNA stand can be terminated with each additional bp, the probability of a DNA strands being sythesised decreases with its lenght. Thus the quantity of a particular DNA stand present decreases with its lenght. This in turn puts a cap on how long a read can go. Once the quantity of DNA synthesis become so low the machine is no longer sensitive enough to detect the signal. I think there is also the problem of separating DNA product that differ by 1 bp... the spacial separation between DNA band becomes less as the band size increases. Setting an upper limit of band resolution (at a particular gel density). And therefore identity of the next bp in a long DNA stand.
Great!!!! very convencing answers. Than you very much
-roniadam-