It is a brief description of restriction fragment length polymorphism enzymes.

1. • Restriction enzymes
• RFLP
Hafiz.M.Zeeshan.Raza
COMSATS University Islamabad,
Sahiwal Campus,
Punjab, Pakistan
hafizraza26@gmail.com

2. Restriction enzymes
1962: “molecular scissors” discovered in in bacteria
These enzymes are found in bacteria and archaea and provide
a defense mechanism against invading viruses. Inside a
prokaryote, the restriction enzymes selectively cut up foreign
DNA in a process called restriction; while host DNA is
protected by a modification enzyme (a methylase) that modifies
the prokaryotic DNA and blocks cleavage.
3,000 enzymes have been identified, around 200 have unique
properties, many are purified and available commercially

3. Methylation

4. Restriction enzymes
• Restriction enzymes are endonucleases (Endo
(inside), nuclease(cuts nucleic acid), which catalyze the
cleavage of the phosphodiester bonds within both
strands of DNA.
• They require Mg+2 for activity and generate a 5
prime (5') phosphate and a 3 prime (3') hydroxyl
group at the point of cleavage.

5. Restriction enzymes
• The distinguishing feature of restriction enzymes is that
they only cut at very specific sequences of bases. This
specific DNA sequence is called recognition sequence.

6. Restriction enzymes
• A restriction enzyme requires a specific double stranded
recognition sequence of nucleotides to cut DNA.
• Recognition sites are usually 4 to 8 base pairs in length.
• Cleavage occurs within or near the site.

7. Restriction enzymes
• The type of bacteria in which the enzyme is found
• The order in which the restriction enzyme was identified and isolated.

8. Many recognition sequences are palindromic. For
example,
5’ GAATTC 3’
3’ CTTAAG 5’
palindromic: read the same in the opposite direction e.g:
Level leveL

9. Sticky and Blunt end cutters
Not all restriction endonucleases cut symmetrically and leave
blunt ends.
Many endonucleases cleave the DNA backbones in positions that are
not directly opposite each other or can make staggered cuts, which
produce single stranded “sticky-ends”
DNA from different sources can be spliced easily because of
these sticky-end overhangs.

10. Example of RE that produce sticky end cutters:

11. Some restriction enzymes cut DNA at opposite base.
They leave blunt ended DNA fragments
These are called blunt end cutters
HaeIII

12. Naturally occurring restriction endonucleases are categorized into four
groups (Types I, II III, and IV) based on their composition and enzyme
cofactor requirements, the nature of their target sequence, and the position
of their DNA cleavage site relative to the target sequence.
All types of enzymes recognize specific short DNA sequences and carry out
the endonucleolytic cleavage of DNA to give specific fragments with
terminal 5'-phosphates.
Types Of Restriction Enzymes

13. • They differ in their recognition sequence, subunit composition, cleavage
position, and cofactor requirements, as summarised below:
• Type I enzymes cleave at sites remote from recognition site; require both
ATP and S-adenosyl-L-methionine to function; multifunctional protein with
both restriction and methylase activities.
• Type II enzymes cleave within or at short specific distances from
recognition site; most require magnesium; single function (restriction)
enzymes independent of methylase.
• Type III enzymes cleave at sites a short distance from recognition site;
require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates
reaction but is not required; exist as part of a complex with a modification
methylase.
• Type IV enzymes target modified DNA, e.g. methylated,
hydroxymethylated and glucosyl-hydroxymethylated DNA.

14. Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a technique in which
organisms may be differentiated by analysis of patterns derived from cleavage of their
DNA.
If two organisms differ in the distance between sites of cleavage of a particular
restriction endonuclease, the length of the fragments produced will differ when the
DNA is digested with a restriction enzyme.
The similarity of the patterns generated can be used to differentiate species (and even
strains) from one another.

15. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments
of the digested DNA sample after they were separated by gel electrophoresis, thus
revealing a unique blotting pattern characteristic to a specific genotype at a specific
locus.
Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP
probes.
Polymorphisms are inherited differences found among the individuals in more than
1% of normal population.

16. Applications of RFLP:
RFLPs can be used in many different settings to accomplish different objectives.
1- RFLPs can be used in paternity cases or criminal cases to determine the source of a
DNA sample. (i.e. it has forensic applications).
2- RFLPs can be used determine the disease status of an individual. (e.g. it can be used in
the detection of mutations particularly known muations)
3- RFLPs can be used to measure recombination rates which can lead to a genetic map
with the distance between RFLP loci measured in centiMorgans.

21. Southern Blotting

22. • Southern blot hybridization is one of the most commonly used
molecular techniques to detect specific DNA sequences using
labeled probes.
• Four steps:
• DNA extraction
• Electrophoresis to separate
• Transfer to membrane
• Use labeled probes, which will hybridize to specific sequence, to identify
sequence of interest

25. Thank you