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DNA molecular testing and
DNA Typing
Genetic testing
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An individual has symptoms or
An individual is at risk of developing a
disease with a family history.
DNA molecular testing:
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A type of testing that focuses on the
molecular nature of mutations associated
with the disease.
Complications
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Many different mutations can cause
symptoms of a single disease.
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BRCA1 and BRCA2 are implicated in the
development of breast and ovarian cancer.
Hundreds of mutations can be found in these
genes; the risk of cancer varies among the
mutations.
General screening and genetic testing are different
(mammograms vs. testing for specific mutations in
the gene).
Genetic testing:
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Prenatal diagnosis: is the fetus at risk?
(amniocentesis or chorionic villus
samples analyzed).
Newborns can be tested for PKU, sickle
cell anemia, Tay-Sachs.
Methods of Genetic Testing
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Restriction Fragment Length
Polymorphism analysis:
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Loss or addition of a RE site is analyzed.
RFLP is a DNA marker.
RFLPs are useful for:
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Mapping the chromosomes.
Finding out different forms of genes/sequences.
RFLPs
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RFLP’s may be changes in the gene of
interest (such as with sickle cell).
Often, RFLP’s are associated with, but not in,
the gene of interest. A RFLP which is very
near the allele of interest will usually indicate
the presence of the allele of interest.
RFLP’s can be used to follow a genetic lineage
(in essence, a specific chromosome) in a
population, and is a useful tool in population
biology.
Different alleles of Hb
Microsatellites and VNTRs as
DNA Markers
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Analysis of “microsatellites” ( short tandem repeats
or STR’s, 2-4 bases repeat), and VNTR’s (Variable
number of tandem repeats, 5- 10’s of bases repeat)
sequences is used in many genetic approaches.
Repeated sequences are often more variable (due to
replication errors and errors in crossing over) than
non repeating sequences, therefore lots of alleles are
generally present in a population.
In other words, two individuals have a higher chance
of genetic differences at STR’s and VNTR’s than at
most sequences in the DNA.
Microsatellites and VNTRs as DNA
Markers
Analysis of Microsatellites and
VNTR’s
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One way of thinking about these analyses is that
this is a specialized RFLP analysis, the power is that
there are lots of alleles in a population, so there is
a high chance that two individuals will be different
in their genotypes (informative).
Two techniques are common in these analyses:
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Southern blot followed by hybridization with a probe that
will detect the sequence (as in RFLP analysis).
PCR with a pair of primers which flank the variable
sequence.
Applications
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Population studies: finding differences in
allele frequencies can identify separate
populations (not interbreeding).
Locating specific genes: associating a specific
VNTR allele with a genetic disease can help
localize the gene to a region of the
chromosome, or trace the allele through a
pedigree.
DNA typing: paternity testing (also useful in
population studies, in animal breeding etc.)
and in forensic analysis.
DNA Typing in
Paternity Testing
• Comparison of
VNTR’s can definitely
exclude an individual
from being the parent
of a child (neither
allele the child has is
present in the alleged
father).
DNA Typing in Paternity Testing
• Proving paternity is more difficult, and relies on statistical
arguments of the probability that the child and the alleged
father are related. Multiple loci (different VNTR’s) must be
examined to provide convincing evidence that the alleged
father is the true father. The same statements (exclusion
versus proof of identity) are true for forensic arguments.
Ethnicity of the accused is a factor: allele frequencies for
VNTR’s are different in different population, be they elk or
human., and often the frequencies (which are the basis of the
statistical arguments) are not known for a specific group.
Finding a Gene: Chromosome
Walking
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Identifying the gene associated with a specific
disease requires years of work.
The first step is to identify the region of the
chromosome the gene is in (pedigree analysis,
identifying breaks in chromosomes which cause
the disease, etc.)
Once the gene has been localized to a region of a
chromosome, is to “walk” along the chromosome.
The walk starts at a sequence known to be
nearby, and continues until the gene of interest is
located.
Isolation of Human Genes
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Positional cloning: Isolation of a gene
associated with a genetic disease on
the basis of its approximate
chromosomal position.
Cystic Fibrosis Gene
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Cystic fibrosis disease is a common lethal
disease inherited as an autosomal recessive
manner.
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Identify RFLP markers linked to the CF gene.
Identify the chromosome on which the CF gene is
located.
Identify the chromosome region on which the CF
gene is located (finer mapping).
Clone the CF gene between the flanking markers.
Identify the CF gene in the cloned DNA.
Identify the defects in the CF gene.
RFLP markers linked to the CF
gene (linkage studies)
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Screen many individuals in CF pedigrees
with a large number of RFLPs.
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Use Southern blot analysis and hybridize
with probes to identify different forms.
Establish a linkage between the markers
and the occurrence of the disease.
Which chromosome?
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Use in situ hybridization, where
chromosomes are spread on a
microscope slide, and hybridized with a
labeled probe, results are analyzed by
autoradiography.
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A 3H-labeled RFLP probe showed that CF
gene is located on chromosome 7.
Which chromosomal region?
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Search other RFLPs located on the chr. 7, to
find ones that are linked to the CF gene.
Again, use the pedigrees and test the DNA for
associated RFLP markers.
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Two closely linked flanking markers (one marker
on each side of the CF gene) were found that are
0.5 map units apart (~500.000 bp).
Their locations were 7q31-q32.
Cloning the CF between
markers
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Chromosome walking technique is used to
walk across the chromosome between the
markers.
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An initial cloned DNA fragment (one of the
flanking markers) is used to begin the walk.
An end piece of this clone is used to screen a
genomic library for clones hybridize with it.
These clones are analyzed by RE mapping to
determine the extent of the overlap.
A new labeled probe is made from right end of the
clone with minimal overlap, the library is screened
again.
• Chromosome walking uses large cloned DNA fragmen
• Clones are isolated from a “library” based on hybridiza
Problems
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End piece of the clone is repetitive DNA, so
that many other chromosomal locations will
give false positive results. So probes should
be unique sequences.
Length of each walk step is limited by the
library. If a gene spans about 500.000 bp, if
the library is a cosmid library (~50.000 bp),
and the average overlap between clones is
about 15.000 bp, then 35.000 x15 = 500.000
bp; 15 steps in the walk is necessary between
flanking markers.
Identifying the CF gene in the
cloned DNA
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Use cloned DNA as probes to hybridize
with other species’ DNA.
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Digest DNA from mouse, hamster or
chicken with RE, analyze fragments by
Southern blotting and hybridization with a
labeled probed.
Select the clone which shows the best
hybridization with other species.
Identifying the CF gene in the
cloned DNA
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Perform a Northern blot; a DNA probe is
hybridized with mRNAs on the blot.
Sequence the selected clone, and look
for regions that may qualify as
promoter regions or exons.
Screen a cDNA library and identify the
clone.
CF gene cDNA is about 6500 bp.
Positional cloning
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Requires knowledge of the gene product
before the gene to be cloned.
Generates transgenic organisms that express
a gene only in certain tissues.
Is when a cDNA has been cloned into a
specific orientation in an expression vector.
Isolates a disease gene based on its
approximate location.
Chromosome walking
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Used to obtain a set of overlapping clones
from a cDNA library.
Used to jump between chromosomal
locations without cloning the intervening
DNA.
Impossible in eukaryotes because of the
amount of interpersed repetitive DNA.
Used to obtain a set of overlapping clones
from a genomic library.
What is the difference between
a pseudogene and a gene.
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A pseudogene is a special type of gene that
contains sequences that hybridize with genes
of other organisms.
A pseudogene is found with CpG islands,
while genes are found next to them.
A pseudogene is stored in heterochromatin,
and is not a functional copy of the gene.
A pseudogene has a sequence resembling a
functional gene, but lacks appropriate
expression signals.
During positional cloning, four
candidate genes are identified.
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What would be the most convincing
evidence?
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A zoo blot
Polymorphisms are present in one of the
genes in affected individuals.
One of them is expressed in the tissue
affected by the disease.
Mutational changes are present in one of
the genes in affected individuals.
Suppose DNA typing is used in
a paternity case.
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How do exclusion results differ from inclusion
results?
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Exclusion results are easier to prove-one needs to
show that the male in question has no alleles in
common with the baby.
Inclusion results require positive identity to be
established and usually testing for alleles at
multiple loci.
Inclusion results require calculation of the relative
odds that an allele came from the accused or from
another person, and requires knowing the
frequencies of VNTR and STR alleles in many
ethnic groups.
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http://www.biology.arizona.edu/human_bio/
activities/blackett/introduction.html
ANSWER THE ACTIVITY QUESTIONS
http://www.biology.arizona.edu/human_bio/
activities/blackett2/overview.html
PERFORM THE ACTIVITES AND ANSWER
QUESTIONS:
Pedigree | Collect data | Paternity testing |
Missing person | RCMP freq. calc.