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DNA/RNA random mutation method?

DNA/RNA random mutation method? - Protocols and Methods Forum

DNA/RNA random mutation method? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-13-2007, 06:50 PM
Z.L. K
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Default DNA/RNA random mutation method?



I would like to generate some random mutations on either DNA/RNA,
however, i won't get it with UV mutagenesis method. Does anyone know
any good method to generate random mutations? Thanks a lot!

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  #2  
Old 06-13-2007, 08:35 PM
DK
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Default DNA/RNA random mutation method?

In article <[Only registered users see links. ].net> , "Z.L. K" <[Only registered users see links. ]> wrote:

Error-prone PCR. Many manufactures sell appropriate polymerases.

DK

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  #3  
Old 06-15-2007, 08:32 AM
Tom Anderson
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Default DNA/RNA random mutation method?

On Wed, 13 Jun 2007, DK wrote:


How about hydroxylamine treatment:

[Only registered users see links. ]

Or EMS:

[Only registered users see links. ]

Use of a mutator strain of _E. coli_ is also an option.


Or, i hear, you can just use the worst normal polymerase you have around
(Taq is fine) and throw in more magnesium, some manganese ions, extra
nucleotides and extra polymerase:

[Only registered users see links. ]

Or, for Quebecois style EP-PCR, some alcohol, bien sur:

[Only registered users see links. ]

tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) [Only registered users see links. ]

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  #4  
Old 06-15-2007, 10:13 PM
DK
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Default DNA/RNA random mutation method?

In article <[Only registered users see links. ].net> , Tom Anderson <[Only registered users see links. ].uk> wrote:

And also drastically reduce concentration of one of the dNTPs.


Yep, that's the ticket. Alcohol is a good way of producing aberrant
progeny :-)

Problem with all of the above is that mutations will not be perfectly
random. Some recent polymerases, OTOH, were tweaked to make
nearly random mistakes.

DK
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  #5  
Old 06-15-2007, 10:35 PM
Joe Doe
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Default DNA/RNA random mutation method?

With the cost of oligos so low I would consider total synthesis with
doped oligos. Probably the only way to control the process well.

Roland
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  #6  
Old 06-16-2007, 10:00 PM
Maximilian Haeussler
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Default DNA/RNA random mutation method?

>Problem with all of the above is that mutations will not be perfectly

RNA viruses achieve a highly random mutations ... I wonder if there a
mutagenetic technique involving transcription to RNA, mutation and
reverse-transcription afterwards...?
Erm, before cliking send, I typed it into google and found this:
[Only registered users see links. ] an RNA-based mutation strategy
based on Qb replicase.

I still wonder that, given that mutagens should be more effective on on RNA
than on DNA, why people don't rather mutate RNA...?

max
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  #7  
Old 06-16-2007, 10:51 PM
WS
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Default DNA/RNA random mutation method?

Oh my dear, they bathed the pol in urea, isopropanol, propan-1-ol, and
butan-1-ol! It must have got quite a hangover! My pol prefers a
delicious Bordeaux Barrique...

For those who want to get access to the paper: The link in PubMed
seems not to work, you'll get an "unknown doi" error message. Check
here directly: [Only registered users see links. ]

Wo

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