Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Polymerase Chain Reaction (PCR)?

Polymerase Chain Reaction (PCR)? - PCR - Polymerase Chain Reaction Forum

Polymerase Chain Reaction (PCR)? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-27-2009, 06:16 AM
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Polymerase Chain Reaction (PCR)?



I need help with these simple questions:

1. PCR consists of 3 major steps: Dissociation, Annealing and synthesis at particular temperatures. What would happen if the temp. was either too high or too low at each of those steps.

2. What would be the consequence if ONE of the primers used had 4 mismatched bases in the middle?

3. We ran PCR for 30 cycles. What would be the consequence of running PCR 1 cycle less? 5 cycles less? 10 cycles less?

4. What would happen if we only used one primer (left or right) in a PCR reaction?

I generally know the answers, I just need to be sure and if I can get thorough and specific answers, that would be great. Thanks!
Reply With Quote
  #2  
Old 10-27-2009, 06:16 AM
Pipette Filler
Points: 2, Level: 1 Points: 2, Level: 1 Points: 2, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default

1. Depends on the step and how drastic the difference but in general if the temps are not correct, those steps can not happen.

2. The primer would not anneal.

3. The consequence would be half as much DNA for each cycle less.

4. You would not amplify a piece of DNA, the primer would still bind and the reaction would still happen but you wouldn't amplify a piece... you'd just get a little bit more of the single piece of DNA.
Reply With Quote
  #3  
Old 10-27-2009, 06:16 AM
Pipette Filler
Points: 2, Level: 1 Points: 2, Level: 1 Points: 2, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default

1. Depends on the step and how drastic the difference but in general if the temps are not correct, those steps can not happen.

2. The primer would not anneal.

3. The consequence would be half as much DNA for each cycle less.

4. You would not amplify a piece of DNA, the primer would still bind and the reaction would still happen but you wouldn't amplify a piece... you'd just get a little bit more of the single piece of DNA.
Reply With Quote
  #4  
Old 10-27-2009, 03:37 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: Polymerase Chain Reaction (PCR)?

Some slightly different answers:

1. Dissociation: Too high, doesn't matter; too low doesn't work (no products)
Annealing: Too high, no annealing and therefore no product.
Too low may work, but your chance of non-specific products goes up a lot
Synthesis: Too high, no products (DNA dissociates)
Too low, fewer to no products, depending on how cold

2. Depends on your annealing temp. Either the primer wont bind, or it'll bind and you'll get PCR products which are mutated to the mis-matched sequence.

3. Depends. Assuming you have enough primer, for every cycles less you loose 1/2 of your yield, for every cycle more you double your yield. 10 cycles less = (1/2)^10 less DNA. For 10 additional cycles you would have 2^10 more DNA - although in a lot of cases you'd run out of primer first, at which point you're extra cycles simple melt, then anneal, your DNA.

4. You'd get single-stranded DNA. This is how sequencing works

Bryan
__________________
[Only registered users see links. ], here at Molecular Station.
Reply With Quote
Reply

Tags
chain , pcr , polymerase , reaction


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
The polymerase chain reaction (PCR) requires __________ and __________ to be Receive PCR - Polymerase Chain Reaction Forum 7 10-21-2009 06:28 AM
The polymerase chain reaction (PCR) requires __________ and __________ to be... Receive PCR - Polymerase Chain Reaction Forum 1 09-02-2009 05:01 AM
The polymerase chain reaction (PCR) requires __________ and __________ to be... Receive PCR - Polymerase Chain Reaction Forum 2 08-28-2009 06:07 PM
PCR World-Everything about Polymerase Chain Reaction peterish PCR - Polymerase Chain Reaction Forum 0 11-27-2008 09:12 PM
Polymerase chain reaction admin Article Discussion 0 03-16-2007 04:31 AM


All times are GMT. The time now is 01:40 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13287 seconds with 16 queries