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Help with sequencing of a 120bp PCR product.


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#1 Wek

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Posted 21 September 2012 - 05:10 PM

I need to demonstrate the sequences of a WT and a mutant DNA gene. However, the PCR fragments are very small, 120bp and 121bp respectively. I have tried sequencing them once but the company said the sequences are not accurate due to the difficulty of working with such a short fragment. Is there any method of getting accurate sequences for these fragments? I have tried looking for gels but haven't been able to find a company that sells gels able to show a 1bp difference.

Also, how can I get a complete and full sequence without any Ns? I seem to have quite a few Ns in my sequences.

TIA

#2 phage434

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Posted 21 September 2012 - 05:46 PM

Easiest would be to clone them into a vector and then sequence the vector. TA cloning of the pcr product would likely be pretty easy.

#3 bob1

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Posted 21 September 2012 - 11:17 PM

You could also try denaturing HPLC.

#4 Wek

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Posted 22 September 2012 - 11:53 AM

Easiest would be to clone them into a vector and then sequence the vector. TA cloning of the pcr product would likely be pretty easy.


I will keep this as a back up option. I was just thinking of just extending the PCR product upstream and downstream the gene to get a larger sequence. It seems to be much easier that to do a blunt ligation to a vector and I don't have much experience with vector ligations.

You could also try denaturing HPLC.


I will have to see if anyone in my institution has equipment and expertise to do this. Never heard of anyone using this technique for sequencing in here.


Anymore suggestions?

#5 Trof

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Posted 22 September 2012 - 01:13 PM

The 120 bp sequence can be sequenced, but it dependes what is the possition of your mutation, if it's near the ends it can be done with no problem, you just need to use very little of template.

Here's the proof, product legth 102 bp.
Attached File  Wildtype fwd.zip   44.26KB   124 downloads
(unzip the file to get electrophoretogram)

You can see only about last 50 bp are readable (first 22 bp is a primer, then the first 30 bases after primer are usually unusable), but that was sufficient for us. With a 120 bp it should be possible to even make a complete read. This is just common ABI sequencing kit (BigDye Terminator v1.1), common sequencer, common polymer and I'm no professional facility. You can send it to your company and ask them how is that possible. If your template is a good quality, length should not be limiting.

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#6 mdfenko

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Posted 25 September 2012 - 08:22 AM

you should be able to separate 1bp difference (for 120 and 121bp) with a sequencing gel (6 or 8% acrylamide with urea) and long.

if you don't have an old sequencing gel apparatus then you should be able to separate them with an automated sequencer that can perform fragment analysis, but then you'll have to attach a fluorescent label.
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#7 Wek

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Posted 25 September 2012 - 09:44 AM

you should be able to separate 1bp difference (for 120 and 121bp) with a sequencing gel (6 or 8% acrylamide with urea) and long.

if you don't have an old sequencing gel apparatus then you should be able to separate them with an automated sequencer that can perform fragment analysis, but then you'll have to attach a fluorescent label.


Is there a difference between a sequencing gel apparatus and a regular electrophoresis apparatus? Could I just use the 6-8% sequencing gel in me electrophoresis apparatus? I am not even sure if I have seen a old sequencing gel apparatus.

Thanks

#8 mdfenko

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Posted 25 September 2012 - 10:45 AM

sequencing gels are longer (40-80cm) than standard protein gels.

you can find an example at labrepco.
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#9 Wek

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Posted 25 September 2012 - 03:02 PM

sequencing gels are longer (40-80cm) than standard protein gels.

you can find an example at labrepco.


Theoretically speaking, could I make a sequencing gel and use it in my large-size electrophoresis apparatus just use it to visualize the 1bp difference? As long as it can show me the 1bp difference I'll be good to go. Eventually I'll get both the sequence and the gel image but I don't want to waste time.

#10 mdfenko

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Posted 26 September 2012 - 05:35 AM

you might be able to but you won't see the same resolution that you would with the proper apparatus. you might be able to see the 1 bp difference within the range of your fragments. try it (you may have to overrun the gel to see the 120 and 121 fragments separate).
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