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PCR questions

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PCR questions

Postby Velinos » Wed Nov 12, 2008 9:39 pm

Sorry if this is the wrong forum, but I thought PCR was closest to genetics.

Doing a review for a quiz tomorrow in biology about PCR since we used it in a lab and these are a few of the questions that I do not understand thoroughly.

The PCR can only be used to amplify genes that have already been cloned and sequenced. Why is this true?

Does this question pertain to genes that have been sequenced in general? I thought the way it worked was:

DNA isolation -> PCR -> electrophoresis -> DNA sequencing -> allelic typing

If sequencing is the 4th phase, and PCR is the 2nd phase, then this question doesn't make sense to me. That's why I think its just a general question? And if this is just a general question, then the answer that I believe is the purpose of having DNA sequenced beforehand (human genome, for instance) then it would be easier for one gene out of the entire genome to produce enough copies that would then be able to determine the nucleotide sequence by biochemical procedures.

If you started a PCR protocol with 10 double stranded molecules of DNA, how many double stranded molecules of product would you have after 5 cycles of PCR?


My answer: 1 = 20, 2 = 40 , 3 = 80, 4 = 160 , 5 =320.
PCR only does multiples of 2 right?

What is the purpose of using DNA polymerase from Thermus aquaticus for PCR rather than a DNA polymerase from a better-characterized bacterium such as e.coli?


Would it be because the e.coli bacterium would not last long? From my book it says that at the beginning of a protocol, Taq DNA polymerase would remain active through at least 30 cycles of PCR. But the thing is that I don't know how long e.coli would last, the only information that I know about it is that its a restriction enzyme and it would be used in electrophoresis.

What is the purpose of including a PCR reaction with no added DNA? Isn't this just a waste of expensive enzyme when we know that without template, you will not get any PCR products?

This is one question that I do not understand. If there is no DNA, then how can a gene be amplified by the PCR and then compared to a template (just as question 1 asks)?

Thanks for any information that may be provided.
Velinos
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Postby canalon » Thu Nov 13, 2008 2:18 am

Moved to molecular biology where the subject belongs...

Help for answers:
1- What are the essential components of a PCR? Which could be linked to sequence?
Now you do not necessarily need a full sequence to do a PCR, but you need some sequence information (sequence of genes located closely, of conserved sequences in the genome, of conserved sequence in the gene,...) to design an iporatnt part of your reaction.

2- Polymerase are enzymes. Enzymes are proteins. Like albuin (in egg whites). Look at what would happen to egg white if you were doing a PCR cycling. Yes, E. coli polymerase would behave the same way... Why? what are the life conditions of E. coli and T. aquaticus. But you could use the E. coli polymerase, it would simply take a bit longer and use way more polymerase

3- Well are you sure there are no DNA? What about the lazy coworker that borrowed your pipettes and reagents and use them without paying attention to aerosols? What about those clouds in your water bottle?
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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Postby biolearner » Thu Nov 27, 2008 9:34 pm

I suggest you the PCR World--everything about polymerase chain reaction (PCR). It's a very organized blog about PCR basis.
http://pcrworld.blogspot.com

Here is the CONTENTS

Polymerase Chain Reaction (Molecular Info)
History of Polymerase chain reaction (PCR)--(1)
History of Polymerase chain reaction (PCR)--(2)
Regular PCR Procedure
Videos and Animations for PCR
Genotyping by PCR
PCR Primer Design Tools
PCR Troubleshooting
About PCR
Introduction to PCR
Polymerase chain reaction--PCR
PCR Animations
PCR, RT-PCR, and Real Time PCR Tutorials
Real Time PCR Protocols
REAL TIME PCR Papers
Variants of PCR (5)
RT PCR Protocols
Web Source of PCR (3)
Special PCR Protocols
Variants of PCR (4)
Variants of PCR (3)
Important Publications at Cell
Important Publications at Nature
Important Publications at Science
Variants of PCR (2)
PCR Manual
PCR SSCP
PCR RFLP
Variants of PCR (1)
AFLP PCR
Alu-PCR
Asymmetric PCR
Colony PCR Protocols
Competitive and/or Quantitative RT-PCR
Degenerate PCR
In Situ PCR
Differential Display PCR
Inverse PCR
Ligation Mediated Suppression PCR
Long PCR Protocols
Methylation Specific PCR
Multiplex PCR
Nested PCR
PCR Elisa
PCR Brochure and Articles
RACE PCR
RAPD PCR
REP-PCR
Tail PCR
Touchdown PCR
Vectorette PCR
Web Source of PCR (3)
Web Source of PCR (2)
Web Source of PCR (1)
http://www.pcrworld.blogspot.com
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