RFLP

By Arjun Elipe
GURU GHASIDAS VISWAVIDYALA
BILASPUR
DEFINITION AND PRINCIPLE:
Restriction Fragment Length
Polymorphism (RFLP) is a widely used
technique in the field of molecular
biology to differentiate the organisms
by analyzing the cleavage patterns of
their DNA using the restriction
enzymes. The technique has found
widespread applications in the field of
forensic science to provide evidences
against the criminals.
PROCESS OF RFLP
*DNA ISOLATION&RE(Restriction Enzymes)
First, the DNA is extracted from the specimens, using
established procedures. In the next step,
the extracted DNA is broken into fragments, using
restriction enzymes. Although there are
several hundred such enzymes, or REs, available
today, the laboratories of most North American
law enforcement agencies and governments (Canada
included) selected one specific RE (called
"HaeIII") in order to achieve uniform results and to
facilitate "networking" of DNA-typing
information
Agarose gel electrophoresis:
After the extracted DNA has been digested by the
enzyme, the various fragments are sorted according to
size, using a technique called agarose gel
electrophoresis, initially used in genetics research and
adapted to forensic use. Agarose gel is a jelly-like
material containing pores through which the DNA
molecules can pass.
The digested DNA samples are loaded into slots at one
end of a flat slab of the gel.
An electric current is applied across the gel causing the
DNA fragments to migrate through the material. The
smaller fragments migrate farther than the larger ones,
to give the end result of an orderly array of fragments
separated by size.
DENATURATION:
In the next step, the DNA fragments are
denatured by soaking the gel in an alkali
solution.(NACL)
The hydrogen bonds holding the two sides
of the double helix of the DNA together are
broken.
With the result that there are now single-
stranded DNA fragments arrayed on the gel
in place of the original double-stranded
fragments
"Southern blotting":
Because the agarose gel is not sufficiently
stable to be used in the rest of the RFLP-
typing procedure, the DNA fragments are
transferred to the surface of a thin nylon
membrane.
This technique, called "Southern blotting" or
"Southern transfer," is named for Edwin
Southern, the scientist who developed it.
When the DNA is fixed to the nylon
membrane, the fragments are ready to be
analyzed
Nucleic acid hybridization.
Hybridization is a process that involves pairing the
single-stranded DNA (nucleic acid) fragments on the
nylon membrane with specific complementary DNA
strands.
The hybridization is carried out with strands of DNA
which have been labelled with a radioactive
isotope, usually an isotope of phosphorus.
These strands are known as DNA probes, so-called
because their base sequences are known and they
are used specifically to bind only to those DNA
strands containing complementary sequences.
Because the probes carry a radioactive label, the
newly hybridized strands can be visualized
X RAY &
Autoradiogra
m
The specimen in
question can then be
compared with known
specimens through their
x-ray images.
If there is a difference in
the patterns between
the DNA from the
suspect individual and
the DNA from the
specimen taken from the
crime scene.
The suspect will be
exonerated. If the
patterns match, the
prosecution can use this
fact as evidence linking
STR
SHORT
TANDEM
REPEATS
 INTRODUCTION
Genomes of eukaryotes are full of repeated DNA
sequences. These sequences are
present in various sizes. They are usually named according
to the length of the core
repeat unit and the number of adjacent repeat units or
complete length of the repeat
region. There might be several hundred to thousand bases
in core repeats of long
repeat units. Those regions of DNA which have short
repeats (2-6 bps in length) are
known as Short Tandem Repeats (STRs). These are highly
polymorphic
microsatellites. The center of the chromosome is
surrounded by STRs.
 Y-STR
These are Short Tandem Repeats present on male
specific Y chromosome. Short arm of Y chromosome
comprises of coding genes which are responsible for
spermatogenesis and other male related functions and
in determination of male sex.
Among unrelated males, these Short Tandem Repeats
are polymorphic. They are inherited from father
(paternal line) and show little change through
generations.
They are mainly used to examine sexual assault
evidence in forensic laboratories. In such cases female
as well as male DNA will be present in vaginal swab
Due to absence of Y
STR in female sample
and presence of Y-
STR in male sample,
the culprit in case of
sexual assault can be
linked to the crime.
Y-STRs are also
useful in non-sexual
assault cases
where the evidence
materials contain
mixed samples of
several males.
Identification of all
males can be done
through Y-STR
testing.
 MINI-STR
Short tandem repeat testing is not successful in
case of highly degraded DNA samples or which are
limited in quality or quantity.
In such situations only partialSTR profile may be
obtained due to drop out of STR alleles. Such
partial DNA profiles do not provide enough
information in forensic cases.
Use of mini-STRs is an alternative approach to such
cases. For mini STR analysis, specially designed
primers are used which target the mini - STR for
amplification.
Mini-STR typing helps in obtaining DNA profile
even from highly degraded samples.
CHARACTERISTICS OF STR SELECTED FOR HUMAN
IDENTIFICATION
i. High discriminating power, usually >0.9, with observed
heterozygosity >70%.
ii. The chromosomal locations chosen should be separate
to make sure that
closely linked loci are not chosen.
iii. The results should be reproducible and robust when
multiplexed with other
markers.
iv. Formation of stutter products (small peaks formed
that are several bases
smaller than STR peaks resulting from PCR process when
short tandem repeat
loci are copied by a DNA polymerase) should be low.
v. Mutation rate should be low.
vi. For analysis of degraded samples alleles with length in
range of 90-500 bps
 Advantages of
STR
i. Multiplexing is possible with a narrow allele size
range.
ii. Allelic dropouts from preferential amplification of
smaller alleles are reduced due to narrow allele size.
iii. STR can generate small PCR products which help
in recovering the information from degraded DNA
samples.
iv. The stutter product formation is reduced as
compared to dinucleotide repeats which help in
interpretation of sample mixtures.
 APPLICATIONS OF STR
1.Detection of
contamination of
tissue samples
2.Detecting Maternal
Cell Contamination
and Fetal Aneuploidy
3.Determining Twin
Zygosity
4.Cancer research
Difference between RFLP and STR