Biochemistry 2280A Study Guide - Final Guide: Bamhi, Dna Ligase, Sticky And Blunt Ends
30 Nov 2015
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11/30/2015 11:54:00 PM
2280 Topic 26 III
To get YFG into E.coli → Plasmid
Plasmid: A piece od DNA that replicates independently from the host
chromosome
Copy number varies (1-100)
Can have a lot of plasmids in the cell
E.coli chrome = 1 copy
small and circular
Plasmid Features → good for cloning
Origin of replication
A selectable marker – usually a gene encoding resistance to an antibiotic
A site into which YFG gene can be inserted → restriction site
Restriction enzymes cut DNA into defined workable units
Help get YFG into plasmid
Restriction enzymes are site specific DNA scissors
Site specific DNA binding proteins that recognize and cleave palindromic DNA
sequences usually of 4,6,8 pairs
Palindromic DNA sequence
Has same sequence on top strand as bottom strand
2 fold symmetry
Restriction enzymes have 2-fold symmetry
Approx 300 commercially available restriction enzymes
Isolated from bacteria where their native role is to act as antiviral agents
Cleavage by EcoRI ** Diagram
Leaves sticky ends with 5’ overhangs (sticky ends)
cut site is asymmetric
Cleavage by Kpnl ** Diagram
Leaves 3’ sticky ends
Ckeavage by Sspl
Cleaves to blunt ends
Some restriction enzymes cut identical sequences but leaves different types
fo ends
BamHI vs NlaIV
find more resources at oneclass.com
find more resources at oneclass.com
restriction enzymes determine cut sites
Some restriction enzymes cut different recognition sequences but leave
identical overhanging ends
BamHI vs BglII
What is the probability that any random 4 bp sequences is a HaeIII site
(GGCC)?
256
Given that the probability of any 6bp sequence bing an Hincll site is
~1/1000, how many Hincll sites are found within the human genome (3
billion bp)
3 million
Restriction enzymes
Affected by conditions (temperature, pH [salt])
Show reaction kinetics
Ligase sticks back together
Reseals compatible sticky ends and much less efficiently blunt ends
requires ATP
Need to have phosphates on ends to put back together
If one of the two has it its fine
Making the recombinant molecules
- Digest ~100ng of YFG and a plasmid with the same restriction enzyme or
one that gives compatible overhanding sequences
- Purify DNA - often by gel electrophoresis
Incubate plasmid + insert YFG in the presence of DNA ligase and ATP
Sticky ends anneal (hybridize)
Ligase seals the ends with covalent Phosphodiester bonds
* How do you get the restriction enzyme sites onto the ends of YFG
Hint has to do with primers
- Introducing YFG into E.coli (transformation)
The process by which cells take up DNA from their environment
Natural property of some bacteria
E.coli must be treated with chemicals to do it well
E.coli does not do it well so needs chemicals – don’t know how this happens
- Plate cells onto agar plates with antibiotic – overnight
Cells with plasmid survive and divide – colonies
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Plasmid: a piece od dna that replicates independently from the host chromosome. Can have a lot of plasmids in the cell. E. coli chrome = 1 copy small and circular. A selectable marker usually a gene encoding resistance to an antibiotic. A site into which yfg gene can be inserted restriction site. Restriction enzymes cut dna into defined workable units. Site specific dna binding proteins that recognize and cleave palindromic dna sequences usually of 4,6,8 pairs. Has same sequence on top strand as bottom strand. Isolated from bacteria where their native role is to act as antiviral agents. Leaves sticky ends with 5" overhangs (sticky ends) cut site is asymmetric. Some restriction enzymes cut identical sequences but leaves different types fo ends. Bamhi vs nlaiv restriction enzymes determine cut sites. Some restriction enzymes cut different recognition sequences but leave identical overhanging ends. Given that the probability of any 6bp sequence bing an hincll site is.
