Transcript DNA fingerprinting

DNA fingerprinting
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Every human carries a unique set of genes (except twins!)
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The order of the base pairs in the sequence of every human varies
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In a single human the DNA sequence in every cell is the same DNA
fingerprinting is based on the two facts:
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there is uniform DNA in an individual
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there is genetic variability between individuals
It is not necessary to catalogue every base pair to arrive at a unique
pattern.
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The original DNA fingerprinting detected unique DNA segments in a
population known as restriction fragment length polymorphisms
(RFLPs).
• A Polymorphism is a sequence of DNA that has many possible
alternatives e.g. different numbers of repeats GC GC GC
• The polymorphic sequences tested in fingerprinting generally come
from areas that do not code. i.e. introns (non coding sequences).
• The name RFLP is derived from the fact that when DNA, extracted
from the cells of an individual, is cut with a restriction enzyme, it
produces a unique pattern of bands when the cut DNA is subjected
to electrophoresis.
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Polymorphic differences in the sequence from individual to
individual give rise to alterations in the sizes of the fragments cut by
restriction enzymes.
• The pattern of restricted DNA fragments (different lengths of DNA)
that appears after electrophoresis can be thought of as a bar code.
• The bands from cells of one individual can be very different from
those of another.
• This is due to the presence of hypervariable regions in the genome.
• Among these hypervariable regions are variable
number tandem repeat sequences (VNTR)
• The number of the VNTRs can vary significantly
from individual to individual
• In humans such sequences are often bordered
by restriction endonuclease sites.
• The fragment sizes resulting from digestion
depend on the number of copies between the
restriction sites
• This gives rise to unique RFLP patterns.
The original procedure used to obtain a DNA fingerprint
1. Isolate genomic DNA
2. cut with restriction enzymes
3. run on a gel
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As humans have more than 3 billion base pairs in their genome , after
electrophoresis all that can be seen is a smear because all the resulting bands
overlap.
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To visualise a fingerprint pattern of a specific VNTR site the DNA fragments must
be detected by probing and hybridisation.
4. After electrophoresis the DNA in the gel is denatured and transferred to a
membrane to make a permanent record.
5. The membrane is then “probed” using a piece of sequence that is complimentary
to the hypervariable region.
6. The binding of the probe is visualised using radioactivity, fluorescence,
conjugated enzyme.
7. The resulting band patterns are a fingerprint.
8. The final DNA fingerprint is built by using several probes (5-10 or more)
simultaneously.
• Today because we have the human DNA
sequence and certain other genome sequences
instead of digesting total genomic DNA and
creating a permanent record on a membrane
that is then probed for variable regions , several
different highly variable regions are amplified
directly by PCR
• FBI uses 22 different regions, RCMP 15 different
regions, paternity tests typically use at least 7
different regions
Interpretations
Applications of DNA fingerprinting
• microbial diagnostics
• genetic diagnostics
• forensic identification
• paternity analysis
• phylogenetic identification
• each person's DNA is as
unique as a fingerprint.
• Blood from the crime
scene evidence, victims
blood, and blood from 2
suspects
• Markers and control DNA
to make sure the the
probes are working
• Suspect 2 can be eliminated
• Is suspect 1 guilty
• Can not be certain. The best we can do is
to estimate the probability that another
person, picked at random, could provide
the same DNA fingerprint.
• The more probes you use, the more
confident you can be that you have caught
the right person.
Major issues with forensic data
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Reliability of Population Data The probability of finding a match
for a particular DNA pattern is found by multiplying the probability
of the separate loci in a particular reference population. The FBI
has developed a database of population statistics for Caucasians,
Blacks, Hispanics and Asians. The density of the population must
also be considered
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Determining a Match This becomes difficult when there are only
small differences in the number of variable number tandem
repeats at a particular locus. Problems are also caused because
of "band shifting" which may occur with differences in the gel or
excess DNA in a well.
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Contamination and Degradation of DNA Samples. Samples
may be contaminated because of bacterial growth in the sample
before it was collected. Old samples may also break down and
give inadmissible results. These samples may have extra bands
or be missing bands.
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Quality Control Practices of Labs. standards for labs are
essential. Absence of standards may lead to poor test quality.
Microbial diagnostics
• Genome-based molecular diagnostics are
used to:
• Detect microbes
• Identify microbes
• Determine antimicrobial susceptibilities
• Perform epidemiologic investigations
Genetic diagnostics
Screening for the sickle-cell gene
• Caused by a mutation in a specific gene
• The only difference between the two genes is the substitution of a T for an A
This
• converts a GAG codon (for Glu) to a GTG codon for Val and
• abolishes a sequence (CTGAGG) recognized and cut by one of the
restriction enzymes.
• When the normal gene (betaA) is digested with
the enzyme and the fragments separated by
electrophoresis, the probe binds to a short
fragment
• However, the enzyme cannot cut the sickle-cell
gene at this site, so the probe attaches to a
much larger fragment
• In this example, a change of a single nucleotide
produced the RFLP. This is a very common
cause of RFLPs and now such polymorphisms
are often referred to as single nucleotide
polymorphisms or SNPs