Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Each of us has the same chemical structure of DNA. But there are millions
of differences in the DNA sequence of base pairs symbolized as A, T, C, G.
This makes the uniqueness among us so that each of us except twins is
different from each other
genetically. DNA sequence
thus can be just like a
fingerprint to identify every
person entirely. But there are
so many base pairs in DNA
sequences that it is hard and
time-consuming to read out all
the sequences. Scientists
have developed many
techniques in order to quicken
the identification process.
Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a difference in
homologous DNA sequences that can be detected by the presence of
fragments of different lengths after digestion of the DNA samples in
question with specific restriction endonucleases. RFLP, as a molecular
marker, is specific to a single clone/restriction enzyme combination. Most
RFLP markers are co-dominant (both alleles in heterozygous sample will
be detected) and highly locus-specific.
Every strand of DNA has pieces that
contain genetic information which
informs an organism's development
(exons) and pieces that, apparently,
supply no relevant genetic
information at all (introns). Although
the introns may seem useless, it has
been found that they contain
repeated sequences of base pairs.
These sequences, called Variable
Number Tandem Repeats (VNTRs),
can contain anywhere from twenty to
one hundred base pairs.
Applications
Forensic Uses
Blood or fragments of tissues such
as hair and skin cells may be left in
the scene of crime. The DNA
fingerprint of these cells from the
scene can be revealed and are
compared with the DNA fingerprint
obtained from criminal suspects. If
somebody's DNA fingerprint
matches with that from the scene
this will be a strong evidence
showing that he has committed the
crime. Also in the case of rapes,
the semen left in the victims'
vagina can also be extracted and
be used to reveal the DNA
sequence which is later compared
with the criminal suspects' DNA
fingerprints so as to find out who
did the rape.
Paternity
A child can inherit most of the DNA fragment
pattern from his parents. By comparing the
DNA fingerprints between the child and the
suspect parents, it is possible to identify who
the parent of the child is.
Southern Blot
A Southern blot is a method routinely used in molecular biology to check for the
presence of a DNA sequence in a DNA sample. Southern blotting combines agarose
gel electrophoresis for size separation of DNA with methods to transfer the sizeseparated DNA to a filter membrane for probe hybridization. The method is named
after its inventor, the British biologist Edwin M. Southern. Other blotting methods (i.e.,
western blot, northern blot, southwestern blot) that employ similar principles, but
using RNA or protein, have later been named in reference to Southern's name. As the
technique was eponymously named, Southern blot should be capitalized, whereas
northern and western blots should not.
Method
Restriction endonucleases are used to cut high-molecular-weight DNA strands into
smaller fragments. The DNA fragments are then electrophoresed on an agarose gel
to separate them by size.
A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or
below, depending on the direction of the transfer) the gel. Pressure is applied evenly
to the gel (either using suction, or by placing a stack of paper towels and a weight on
top of the membrane and gel), to ensure good and even contact between gel and
membrane. Buffer transfer by capillary action from a region of high water potential to
a region of low water potential (usually filter paper and paper tissues) is then used to
move the DNA from the gel on to the membrane; ion exchange interactions bind the
DNA to the membrane due to the negative charge of the DNA and positive charge of
the membrane.
The membrane is then exposed to high temperature (60 to 100 C) (in the
case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to
permanently and covalently crosslink the DNA to the membrane.
The membrane is then exposed to a hybridization probe. The probe DNA is
labelled so that it can be detected, usually by incorporating radioactivity or
tagging the molecule with a fluorescent or chromogenic dye. In some cases,
the hybridization probe may be made from RNA, rather than DNA. To ensure
the specificity of the binding of the probe to the sample DNA, most common
hybridization methods use salmon testes (sperm) DNA for blocking of the
membrane
.
After hybridization, excess probe is washed from the membrane, and the
pattern of hybridization is visualized on X-ray film by autoradiography in the
case of a radioactive or fluorescent probe, or by development of color on the
membrane if a chromogenic detection method is used.
Result
Hybridization of the probe to a specific DNA fragment on the filter membrane
indicates that this fragment contains DNA sequence that is complementary to
the probe.
The transfer step
of the DNA from
the
electrophoresis
gel to a membrane
permits easy
binding of the
labeled
hybridization
probe to the sizefractionated DNA.
It also allows for
the fixation of the
target-probe
hybrids, required
for analysis by
autoradiography
or other detection
methods.
Teacher Key
The High Rise Killer
DNA Marker Unknown
1
115 bp
109 bp
103 bp
95 bp
84 bp
75 bp
62 bp
54 bp
43 bp
30 bp
Victim
Suspect #1
Suspect #2
Student Worksheet
The High Rise Killer
DNA Marker Unknown
1
115 bp
109 bp
103 bp
95 bp
84 bp
75 bp
62 bp
54 bp
43 bp
30 bp
Victim
Suspect #1
Suspect #2