Download 2015 teacher-prof dev- restriction enzyme lecture

Restriction Enzyme Digest
October 8, 2015
Restriction Enzymes
Restriction Enzymes
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Also known as restriction endonucleases
Scan the DNA sequence
Find a very specific set of nucleotides
Make a specific cut
Used to construct recombinant DNA plasmids
Palindromes in DNA sequences
5
’
3’
3’
5
’
Genetic palindromes
are similar to verbal
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
sequence is identical
on both strands.
Restriction enzymes recognize and
make a cut within specific
palindromic sequences, known as
restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.
Restriction Endonuclease Types
Type I- multi-subunit, both endonuclease and
methylase activities, cleave at random up to
1000 bp from recognition sequence
Type II- most single subunit, cleave DNA within
recognition sequence
Type III- multi-subunit, endonuclease and
methylase about 25 bp from recognition
sequence
Hae III
HaeIII is a restriction enzyme that
searches the DNA molecule until it finds
this sequence of four nitrogen bases.
5’ TGACGGGTTCGAGGCCAG
3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site is found Hae III will
cleave the DNA at that site
5’ TGACGGGTTCGAGGCCAG
3’
3’ ACTGCCCAAGGTCCGGTC
5’
These cuts produce
“blunt ends”
5’ TGACGGGTTCGAGG
3’ ACTGCCCAAGGTCC
CCAG 3’
GGTC 5’
The names for restriction enzymes come from:
• the type of bacteria in which the enzyme is found
• the order in which the restriction enzyme was identified
and isolated.
EcoRI for example
R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.
“blunt ends” and “sticky ends”
Hae III produced a “blunt end”?
EcoRI makes a staggered cut and produces a
“sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA
G 5’
More examples of restriction sites of restriction
enzymes with their cut sites
Hind III: 5’ AAGCTT 3’
3’ TTCGAA 5’
Bam HI: 5’ GGATCC 3’
3’ CCTAGG 5’
Alu I: 5’ AGCT 3’
3’ TCGA 5’
Separating Restriction Fragments
Our digests:
…
Run digest on gel to check fragment sizes
• miR-29c construct ~ 5kB plasmid
– BglII/EcoRI digest ~250bp fragment
• PRKCBP1 construct ~9kB plasmid
– AsiSI/BglII digest ~1.8kB fragment
Uses of restriction enzyme analysis- forensics
http://science.howstuff
works.com/dnaprofiling1.htm
Uses of restriction enzyme analysisMedical diagnosis
http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
Uses of restriction enzyme analysisInheritance and genetic counseling
http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
Sanger sequencing:
https://en.wikipedia.org/wiki/Sanger_sequencing#/media/File:Sanger-sequencing.svg
Cost to sequence DNA has dropped dramatically
Hayden, Nature 2014
Next-generation sequencing:
Metzker, Nat Rev Gen 2010