Download restriction enzymes

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restriction enzymes
R. Ward: Spring 2001
•Restriction Enzymes (endonucleases): molecular scissors
that cut DNA
•Properties of widely used Type II restriction enzymes:
•recognize a single sequence of bases in dsDNA,
usually symetrical (palindromic)
•cleave both strands, generally within the
recognition sequences
•leaving “blunt” or “sticky” ends
•leave a 3'-hydroxyl on one side of the cut and a
5'-phosphate on the other side of each strand
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Examples of
restriction enzymes
R. Ward: Spring 2001
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Palindromic sequences
Axis of symmetry
5’ – NNNNNNNNNGGATCCNNNNNNNNNN – 3’
3’ – NNNNNNNNNCCTAGGNNNNNNNNNN –5’
5’
G
3’
C
G
C
A
T
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T
A
R. Ward: Spring 2001
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R. Ward: Spring 2001
•Gene cloning = replication of a target sequence of DNA
•insert target sequence into an easily replicated vector
•insert the vector into a single bacteria (transformation)
•allow the bacteria to amplify
•vector has sequences that enable coordinated replication of the recombinant vector DNA
•DNA Cloning: Vectors
•Properties of useful vectors:
•Vector DNA can be introduced into a bacteria
•Vector contains a replication origin so it can replicate inside a host cell
•vector contains antibiotic resistance gene or other selectable marker gene that allows easy identification of transformed bacterial colonies
•Cloning vectors
•Plasmid: insert DNA max size= 15 kb; autonomous replication; contains antibiotic resistance genes
•Bacteriophage lambda: insert = 25 kb; recombinant DNA packaged into phage particles used to infect E. coli
•BACs: bacteria artificial chromosomes insert size 100-500 kb
•YACs: yeast articifical chromosomes 250-1000kb (1 mb)
•cloning
•generate fragments with a restriction enzyme. Sources can be whole DNA sample (genomic), or DNA generated from RNA of particular
tissue
•mix with linearized (restricted) plasmid (cut with same enzyme
•ligate
•get two products: plasmid with insert, plasmid only
•have site in middle of gene whose function is disrupted upon insertion
•bacterial amplification
•transform bacteria with plasmid/insert construct
•grow (plate) single cell dervied colonies on media carrying selectable agent (antiotic, usually)
•grow single colony derived bacterial strains that survive the antibiotic and exhibit evidence of insertion
•maintain as colony on plate or as frozen solution. “Library” is a collection of clones
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R. Ward: Spring 2001
•Cloning vectors
•Plasmid: insert DNA max size= 15 kb; autonomous replication; contains
antibiotic resistance genes
•Bacteriophage lambda: insert = 25 kb; recombinant DNA packaged into
phage particles used to infect E. coli
•BACs: bacteria artificial chromosomes insert size 100-500 kb
•YACs: yeast articifical chromosomes 250-1000kb (1 mb)
•cloning
•generate fragments with a restriction enzyme. Sources can be whole DNA sample
(genomic), or DNA generated from RNA of particular tissue
•mix with linearized (restricted) plasmid (cut with same enzyme
•ligate
•get two products: plasmid with insert, plasmid only
•have site in middle of gene whose function is disrupted upon insertion
•transform bacteria with plasmid/insert construct
•grow (plate) single cell dervied colonies on media carrying selectable agent
(antiotic, usually)
•grow single colony derived bacterial strains that survive the antibiotic and exhibit
evidence of insertion
•maintain as colony on plate or as frozen solution. “Library” is a collection of
clones
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Main point on BACs and YACs is the large size insert they
can carry
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Purifying insert involves separating plasmid from bacterial chromosomal DNA,
followed by restriction with original cloning enzyme, then separation of plasmid
(or other vector) from insert
Methods of separation include electrophoresis
You can also use PCR to amplify the insert directly from bacterial colonies or
purified vectors by using the DNA sequence info flanking the vector cloning site
to design forward and reverse primers.
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Libraries: Collections of DNA clones
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vGenomic
libraries are made from “genomic”
DNA directly from the plant’s DNA
vcDNA libraries are made from DNA fragments
that are created with reverse transcriptase from
mRNA isolated from the plant
vComplete libraries hypothetically contain one or
more copies of every sequence from the source
tissue.
vLibraries maintained as bacterial or yeast colonies
R. Ward: Spring 2001
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Subcloning
R. Ward: Spring 2001
Lambda (Phage) sub-libraries of individual YAC or BAC inserts enable
manipulation of 20kb or so subsequences from that YAC/BAC insert
Each sub-library can then be sub-cloned further into plasmids for even higher
resolution.
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cDNA
R. Ward: Spring 2001
Big message here is that mRNA can be converted into DNA,
called cDNA
cDNA inserts do not have promoter or introns from the
original genomic locus that the mRNA came from
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