"Subclassing and Composition – A Pythonic Tour of Trade-Offs", Hynek Schlawack
VNTR and RFLP
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2. CONTENTS:
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RFLP
What is RFLP…
Method of DNA analysis by RFLP
Applications of RFLP
VNTR
What is VNTR…
VNTR Structure and allelic variation
Use of VNTR in analyzing genetics
VNTR inheritance
General method of VNTR
Applications of VNTR
3. WHAT IS RFLP…
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.
4. WHAT IS RFLP…
Most
RFLP markers are co-dominant (both alleles in
heterozygous sample will be detected) and highly
locus-specific.
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.
5. METHOD
OF DNA ANALYSIS BY RFLP
The method of analysis of DNA by RFLP
involves the following steps:
1- In the first step fragmentation of a sample of
DNA is done by a restriction enzyme, which can
recognize and cut DNA wherever a specific short
sequence occurs, in a process known as a
restriction digest.
6. 2- The resulting DNA fragments are then separated by
length through a process known as agarose gel
electrophoresis.
3- Then transferred to a membrane via the Southern blot
procedure.
4- Hybridization of the membrane to a labeled DNA
probe will done and then determines the length of the
fragments which are complementary to the probe.
7. 5- Then we will observe the fragments of different
length.
An RFLP occurs when the length of a detected
fragment varies between individuals. Each fragment
length is considered an allele, and can be used in
genetic analysis.
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9. APPLICATIONS OF RFLP
RFLPs can be used in many different
settings to accomplish different objectives as:
1- Paternity case:
RFLPs can be used in paternity cases or criminal
cases to determine the source of a DNA sample.
(i.e. it has forensic applications).
10. 2- Detection of Recombination rate:
RFLPs can be used to measure recombination
rates which can lead to a genetic map with the
distance between RFLP loci.
3- Agriculture:
Direct method for selecting desirable genes such
as disease resistance
11. 4- Genetic mapping:
Determine disease status of an individual, i.e.
Huntington’s chorea
Cystic fibrosis
Sickle cell anemia
5- Genetic Counseling:
Very important when discussing results with patients
or parents who use this technology to have children
who are free of genetic disease.
12. 6- Disease detection:
RFLPs can be used determine the disease status of an
individual. (e.g. it can be used in the detection of
particular mutations)
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14. WHAT IS VNTR…
A Variable Number Tandem
Repeat (or VNTR) is a location in a genome where a
short nucleotide sequence is organized as a tandem
repeat. These can be found on many chromosomes, and
often show variations in length between individuals.
Each variant acts as an inherited allele, allowing them to
be used for personal or parental identification. The size
of repeat is about 7 to few tens of base pairs long.
15. VNTR STRUCTURE AND ALLELIC VARIATION
The rectangular blocks represent each of the
repeated DNA sequences at a particular VNTR location.
The repeats are tandem - they are clustered together and
oriented in the same direction. Individual repeats can be
removed from (or added to) the VNTR
via recombination or replication errors.
The VNTR blocks can be extracted
with restriction enzymes and analyzed by RFLP, or
amplified by the polymerase chain reaction (PCR)
technique and their size determined by gel
electrophoresis.
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17. USE OF VNTR IN ANALYZING GENETICS
VNTRs were an important source of RFLP genetic
markers used in linkage analysis (mapping) of genomes.
When removed from surrounding DNA by the PCR or RFLP
methods, and their size determined by gel electrophoresis
or Southern blotting, they produce a pattern of bands unique
to each individual. When tested with a group of independent
VNTR markers, the likelihood of two unrelated individuals
having the same allelic pattern.
18. VNTR INHERITANCE
In analyzing VNTR data, two basic genetic principles can
be used:
1.
Identity Matching- both VNTR alleles from a
specific location must match. If two samples are from
the same individual, they must show the same allele
pattern.
2. Inheritance Matching- the VNTR alleles must
follow the rules of inheritance. In matching an
individual with his parents or children, a person must
have an allele that matches one from each parent. If the
relationship is more distant, such as a grandparent or
sibling, then matches must be consistent with the
degree of relatedness.
19. GENERAL METHOD OF VNTR
In first step we use restriction enzymes to cut the VNTR
blocks from its specific sides and analyzed by RFLP
In second step we amplified PCR techniques
In last step we run it on gel electrophoresis which also
determined its length
