Download Restriction Enzymes

Lab.
Restriction Enzymes
Objectives:
1- Introduce the students to digest genomic DNA by restriction
endonucleases
2- Observe the results of digestion on agarose gel electrophoresis.
Introduction:
Restriction enzymes (also known as restriction endonucleases) found in bacteria (and
harvested from them for use) and cut DNA up at specific sequences in the genome.
For example, the commonly used restriction endonuclease EcoRI recognizes every
point in DNA with the sequence GAATTC, and cuts at the point between the Guanine
and Adenine. Interestingly, the recognition sequences for most restriction
endonucleases are genetic palindromic, e.g., the sequence reads exactly the same
backwards on the complementary strand.
Examples of restriction enzymes include:
Enzyme
Source
Recognition Sequence
Cut
5'GAATTC
3'CTTAAG
5'---G
AATTC---3'
3'---CTTAA
G---5'
5'CCWGG
3'GGWCC
5'--CCWGG---3'
3'---GGWCC
---5'
BamHI Bacillus amyloliquefaciens 3'CCTAGG
5'GGATCC
5'---G
GATCC---3'
3'---CCTAG
G---5'
HindIII Haemophilus influenzae
5'AAGCTT
3'TTCGAA
5'---A
AGCTT---3'
3'---TTCGA
A---5'
TaqI
Thermus aquaticus
5'TCGA
3'AGCT
5'---T
CGA---3'
3'---AGC
T---5'
HinfI
Haemophilus influenzae
5'GANTC
3'CTNAG
5'---G
ANTC---3'
3'---CTNA
G---5'
Sau3A
Staphylococcus aureus
5'GATC
3'CTAG
5'--GATC---3'
3'---CTAG
---5'
PovII*
Proteus vulgaris
5'CAGCTG
3'GTCGAC
5'---CAG
3'---GTC
CTG---3'
GAC---5'
SmaI*
Serratia marcescens
5'CCCGGG
3'GGGCCC
5'---CCC
3'---GGG
GGG---3'
CCC---5'
HaeIII* Haemophilus aegyptius
5'GGCC
3'CCGG
5'---GG
3'---CC
EcoRV* Escherichia coli
5'GATATC
3'CTATAG
5'---GAT
3'---CTA
EcoRI
Escherichia coli
EcoRII Escherichia coli
* = blunt ends
N = C or G or T or A
W = A or T
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CC---3'
GG---5'
ATC---3'
TAG---5'
Restriction enzymes are part of a bacteria's ''immune'' system. These are enzymes
that cut DNA at specific sites (typically a four or a 6 base-pair sequence). Bacterial
DNA is modified to be protected by methylation while foreign DNA, such as
incoming viruses, are not. Usually, organisms that make restriction enzymes also
make a companion modification enzyme ( DNA methyltransferase) that protects their
own DNA from cleavage. These enzymes recognize the same DNA sequence as the
restriction enzyme they accompany, but instead of cleaving the sequence,
they disguise it by methylating one of the bases in each DNA strand.
Thus, we can take a long piece of DNA and cut it with a restriction enzyme,
generating numerous fragments. Even a single-base change will destroy a restriction
enzyme target site. Likewise, even if a site is the same in two molecules, the lenght of
DAN sequence between them may change. Thus if two DNA molecules differ in
sequence, they likely have different lengths for the fragments produced following
treatment with restriction enzymes.
Typical digestions included a unit of enzyme per microgram of starting DNA, and one
enzyme unit usually (depending on the supplier) is defined as the amount of enzyme
needed to completely digest one microgram of double-stranded DNA in one hour at
the appropriate temperature. These reactions usually are incubated for 1-3 hours, to
insure complete digestion, at the optimal temperature for enzyme activity, typically
37degC.
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The Digests:
1. Prepare the reaction for restriction digestion by adding the following reagents in the
order listed to a microcentrifuge tube:
Example:MTHFR PCR products
Components of the reaction
Volum
PCR product
14 µl
2 µl
2 µl
2 µl
20 µl
dH20
Restriction buffer 10x
Hinf1 enzyme (1-10 units
Total volume
per ug DNA)
2. Gently mix by pipetting and incubate the reaction at the appropriate temperature
(typically 37degC) for 1-3 hours or overnight.
3. Inactivate the enzyme(s) by heating at 70-100degC for 10 minutes.
4. Run on the agarose gel 2%, o freeze the samples until you have time to run.
5. Record your observation .
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