Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum > DNA Forum
Register Search Today's Posts Mark Forums Read

DNA Forum Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


Plasmid PCR question...

Plasmid PCR question... - DNA Forum

Plasmid PCR question... - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-30-2010, 05:09 PM
Pipette Filler
Points: 15, Level: 1 Points: 15, Level: 1 Points: 15, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Angry Plasmid PCR question...



I am a biochemist working with a bunch of physicists, and an interesting point came up the other day while discussing PCR in plasmids.

At the end of the extension, you are left with a nicked piece of DNA. The nick appears between the beginning of the primer, and the last nucleotide added to the circle. During the next cycle, heating the dsDNA to 95 degrees melts the two strands apart. Why does the nicked strand not linearize? How is it that you end up with circular product?

The next round of PCR should start by adding a primer and extending the chain over the nick to form circular product. How is the polymerase able to reconcile this nick, possibly linear at this point? How does it find where to start replicating the chain?

I would never have thought about this without them asking me, and tech consultants from Stratagene have been unable to answer this question... I am hoping someone on here can lend insight.

Is it due to DNA persistence length? Stacking energies? I have no idea.
Reply With Quote
  #2  
Old 08-31-2010, 05:06 AM
Banned
Points: 16, Level: 1 Points: 16, Level: 1 Points: 16, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2010
Posts: 7
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Plasmid PCR question...

Hope you can have a better idea then!
Reply With Quote
  #3  
Old 11-17-2010, 03:18 PM
Pipette Filler
Points: 25, Level: 1 Points: 25, Level: 1 Points: 25, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2010
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Plasmid PCR question...

I also have the same question (I'm the physicist working with biochemists!). I'm trying to obtain linear ssDNA and was thinking about doing PCR on a single-stranded plasmid with only one primer. After many cycles, would this give me mostly circular DNA or linear?
Reply With Quote
  #4  
Old 04-14-2011, 10:21 AM
Pipette Filler
Points: 44, Level: 1 Points: 44, Level: 1 Points: 44, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2011
Location: Kobe, Japan
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Plasmid PCR question...

Hope all of you have a better idea soon
Reply With Quote
  #5  
Old 04-14-2011, 10:02 PM
Pipette Filler
Points: 45, Level: 1 Points: 45, Level: 1 Points: 45, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2011
Location: U.S.
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Plasmid PCR question...

Asking why you would end up with a circle is like asking why the primer adheres to your template in the first place. You have all these fragments moving around, and when one of the "nicked" ends in the circle adheres to one side of the strand you are trying to insert, at that moment, you do have a linear piece. But why did they adhere in the first place? Because they are complementary. Similarly, the other end of your circular DNA complements the other end of the insert. So they are likely to combine, in fact, they would prefer it (if given the choice). Their close proximity enhances the likelihood for recombination even more. This is why you have to look for dimers, etc, when choosing a primer, because you don't want them to adhere to themselves!

I don't have any hard evidence, but I would say far and away the majority of your amplicons are going to be circular. I really can't imagine ending up with a lot of linear strands.
Reply With Quote
The Following User Says Thank You to 4N6addict For This Useful Post:
admin (04-15-2011)
Reply

Tags
nicked dna , pcr , physics , plasmid , question


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
Mutation to Increase Plasmid Copy Number Question rbb147 DNA Forum 0 06-27-2010 06:06 PM
Plasmid DNA question? Any help appreciated.? everythings_magic DNA Techniques 1 11-04-2008 03:56 AM
could i ask you a question about suicide plasmid? yuyanxiong Microbiology Forum 0 06-02-2008 10:17 AM
Reuse of Plasmid with Insert from Agrobacterium cells molbio123 DNA Techniques 2 08-05-2007 05:47 PM
yeast plasmid preps johnspar@earthlink.net Yeast Forum 0 02-20-2004 02:13 AM


All times are GMT. The time now is 05:28 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 0.13635 seconds with 16 queries