Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > DNA Techniques
Register Search Today's Posts Mark Forums Read

DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


Restriction Enzyme Digestion

Restriction Enzyme Digestion - DNA Techniques

Restriction Enzyme Digestion - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-05-2006, 04:35 PM
Pipette Filler
Points: 1,887, Level: 26 Points: 1,887, Level: 26 Points: 1,887, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2006
Posts: 19
Thanks: 0
Thanked 0 Times in 0 Posts
Talking Restriction Enzyme Digestion



Hi to all,

I am currently attempting to do digestion of my plasmids now.

However, I am wondering on how the enzymes really work since my basic in Biology is weak.

The enzymes have a specific site to cleave the genes in the plasmids at the multicloning site. The enzymes are resembled in the products' website with a 5' to 3' and 3' to 5'. Therefore, it seems like they are a string of DNA. Since DNA in plasmids are in double helical forms, how does the enzyme actually cut the plasmids and what will become of the plasmids after that? open circular and nicked are still in double helical form?

Thank you.
Reply With Quote
  #2  
Old 10-05-2006, 04:59 PM
Graduate Student
Points: 2,923, Level: 35 Points: 2,923, Level: 35 Points: 2,923, Level: 35
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2006
Location: Ithaca, NY
Posts: 145
Thanks: 0
Thanked 17 Times in 12 Posts
Default Re: Restriction Enzyme Digestion

Think of double-helix DNA as two sticky strings (say, one blue and one red) twisted together. To make a plasmid, take the twisted string and tie the ends of the red strings together, then tie the ends of the blue string together. You'll have a circle made up of two circular pieces of string that are twisted and stuck together.

Supercoiled plasmid occurs when the red and blue are twisted together so tightly that they start to kink up (Seriously, take a piece of string and twist it, you'll see what I mean.) If a plasmid is nicked, imagine untying the knot in the red string. Now it can untwist around from the blue string and release some of the kinks. It's still double-helical, since the two strings are stuck together (modeling the base pair bonding), but it's in an "open" or "relaxed" conformation.

Here's where my model breaks down: each of the strings has a direction, and the two strings' directions are opposite. That's the 5'/3' designation.

Enzymes usually recognize a palendromic DNA sequence (for example, EcoRI cuts 5'-G'AATTC-3'. Because it's palendromic, when you have the double stranded DNA, you have the same sequence reading either way:
Code:
5'-G'AATT C-3'
3'-C TTAA'G-5'
(if you take your monitor and flip it upside down, you'll see that this sequence looks the same either way you look at it)

So, the enzyme recognizes the double-stranded sequence, and cuts each strand where I've put the tick mark, giving you a linear plasmid with the ends:

Code:
5'-G         AATTC-3'
3'-CTTAA         G-5'
The string is still twisted and stuck together, but it's back to being a string rather than a circle.

Does this help at all? Or make any sense to you? Or was this not your question?
Reply With Quote
  #3  
Old 10-05-2006, 05:48 PM
moleculardude's Avatar
M.D/Ph.D
 
Join Date: Jul 2006
Location: Melbourne Australia
Posts: 423
Thanks: 5
Thanked 10 Times in 8 Posts
Default Re: Restriction Enzyme Digestion

Great post Kmunson. I learned a few things from that too!

Yes open circular and nicked are both double helical. The enzyme only affects the ends of the DNA molecule, not the structure.

Although, I am not sure what happens to the single stranded DNA ends. I guess they are not double-helical as they are single stranded.

See Kmunsons post above. The unpaired nucleotides on the ends as you see will not be double helical as they are unpaired ( hence no double helix).

Actually the only one restriction cut that has double helix ends is the BLUNT cut where it is like this:

GACTACC
CTGATGG

after cutting

GAC TACC
CTG ATGG

Reply With Quote
  #4  
Old 10-05-2006, 06:40 PM
Graduate Student
Points: 2,923, Level: 35 Points: 2,923, Level: 35 Points: 2,923, Level: 35
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2006
Location: Ithaca, NY
Posts: 145
Thanks: 0
Thanked 17 Times in 12 Posts
Default Re: Restriction Enzyme Digestion

Quote:
The unpaired nucleotides on the ends as you see will not be double helical as they are unpaired ( hence no double helix).
Right. Sorry I missed that -- the EcoRI ends are single-stranded DNA, called "sticky ends" since they want to base pair with a complementary single strand...
Reply With Quote
  #5  
Old 10-06-2006, 06:34 PM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default Re: Restriction Enzyme Digestion

Hey Kmunson,

I never thought of that either until moleculardude pointed it out.

thanks moleculardude
Reply With Quote
  #6  
Old 10-15-2006, 10:09 AM
Pipette Filler
Points: 1,887, Level: 26 Points: 1,887, Level: 26 Points: 1,887, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2006
Posts: 19
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Restriction Enzyme Digestion

kmunson779 and molecular dude ..

Thanks a lot. that helped clear some haze in my mind.
Reply With Quote
Reply

Tags
digestion , enzyme , restriction


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
problems with restriction digestion chemophoto@gmail.com Protocols and Methods Forum 7 01-04-2012 01:00 PM
Restriction Enzyme Digestion Protocol admin DNA Techniques 1 04-13-2011 06:11 AM
Restriction enzyme digestion giusygiusy Transfection Forum 2 01-24-2010 09:42 PM
gel electrophoresis restriction enzyme digestion HELP!? Chris M DNA Techniques 0 10-26-2008 12:20 PM
Restriction Enzyme Digestion admin DNA Techniques 0 08-02-2006 09:44 AM


All times are GMT. The time now is 10:28 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14640 seconds with 16 queries