Restriction enzymes

1. MOLECULAR BIOLOGY
BCHM 415

2. • Restriction enzymes (RE) are enzymes that recognize specific short (4-6)
nucleotide sequences in double stranded DNA and cleave specific sites on those
sequences.
• The specific site is called a restriction site.
• Restriction enzymes are also called biological/molecular scissors.
•These enzymes were discovered in bacteria.
•They help the bacteria destroy viral DNA (bacteria get viruses too!)
A bacterium is immune to its own restriction enzymes, even if it has the target
sequences ordinarily targeted by them. Why? 2
Restriction Enzymes

3. Restriction Sequences are usually palindromic
(Words that read the same forwards as backwards)
Hannah
Level
Madam
hannaH
leveL
madaM

4. Palindromes in DNA sequences
A palindromic sequence in
DNA is one in which the 5’ to
3’ base pair sequence is
identical on both strands (the
5’ and 3’ ends refers to the
chemical structure of the
DNA).

5. • EcoRI:
Isolated from E.coli strain RY13.
I indicates thatit was the first enzyme of that type isolatedfrom E. coli RY13.
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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.

6. RE
name
Origin
Restriction site
EcoRI Escherichia coli
BamHI Bacillus amyloliquefaciens H
HindIII Haemophilus influenza RD
HaeIII Haemophilus aegyptius
AluI Arthrobacter luteus
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7. Blunt end
Sticky end
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Products of restriction digestion have either sticky or blunt ends

8. Sticky ends are useful
DNA fragments with
complimentary sticky ends
can be combined to create
new molecules, and this
allows the creation and
manipulation of DNA
sequences from different
sources.
Think about how this could be
used and abused in the
medical field

9. A restriction enzyme first scans the entire length of the DNA.
It the binds to the DNA molecule when it recognizes a specific sequence.
It finally makes one cut in each of the sugar phosphate backbones of the double
helix – by hydrolysing the phosphodiester bond (specifically between the 3’ O
atom and the central P atom).
(Scan Recognize Cut)
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10. Example:
If EcoRI recognizes 5’-GAATTC-3’ sequence and cuts
between G and A:
(a) how many paired fragments will be obtained if the
double stranded DNA strand below is digested with this
enzyme?
(b) Write the sequences of each of the fragments.
(b) Estimate the size of each fragment on agarose gel.
5’-TCCAGCTGGACGAATTCTTCAGATGAATTCAAA-3’
3’-AGGTCGACCTGCTTAAGAAGTCTACTTAAGTTT-5’

11. Uses of restriction enzymes
1. Generation of restriction map.

12. 2. Production of Recombinant DNA

13. 3. Analysis of Restriction Fragment Length Polymorphism (RFLP).

14. Questions???

15. • Is a tool to study variations among individuals (humans and other species).
• This technique able to differentiate minor nucleotide sequence variations in
homologous fragments of DNA
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16. DNA
Extraction
PCR for the
region thatyou
want to dothe
study on
Incubation of
the DNA with
RE
Agarose gel to
visualized your
results
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17. • Geneticdisease analysis as application.
MstII restriction stite:
‘5-CCTNAGG-3’
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18. 18

19. • Restriction of genomicDNA.
• Genomic DNA or DNA fragments obtainedfollowing PCR incubatedwith ER under
appropriate experimentalconditions.
• Resulted restrictionfragmentscan be separatedon agarose gel electrophoresis by size.
• In this experiment restriction of genomic DNA will be done using MstII, which cut the DNA at
‘5-CCTTAGG-3’
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20. 1. Label a clean microcentrifuge tube,and add the following:
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2. Add MstII (3 U for each one µg DNA) and incubate the reaction mixturefor 20 min at 37 °C
in an incubator.
3. Stop the reactionby adding 0.5 µl of 0.5 M EDTA.
4. Prepare it for agarose gel electrophoresisby adding 5 µl of gel loading buffer
Component Volume (µl)
DNA solution (0.5 µg/µl) 1
10X restriction buffer 2
NaCl solution 1
Water 16