DNA molecular testing and DNA Typing

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DNA molecular testing and DNA Typing
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Genetic testing

• An individual has symptoms or
• An individual is at risk of developing a disease
  with a family history.
• DNA molecular testing
• A type of testing that focuses on the molecular
  nature of mutations associated with the disease.

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Complications

• Many different mutations can cause symptoms of a
  single disease.
• 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).

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Genetic testing

• Prenatal diagnosis is the fetus at risk?
  (amniocentesis or chorionic villus samples
  analyzed).
• Newborns can be tested for PKU, sickle cell
  anemia, Tay-Sachs.

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Methods of Genetic Testing

• Restriction Fragment Length Polymorphism
  analysis
• Loss or addition of a RE site is analyzed.
• RFLP is a DNA marker.
• RFLPs are useful for
• Mapping the chromosomes.
• Finding out different forms of genes/sequences.

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RFLPs

• RFLPs may be changes in the gene of interest
  (such as with sickle cell).
• Often, RFLPs 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.
• RFLPs can be used to follow a genetic lineage
  (in essence, a specific chromosome) in a
  population, and is a useful tool in population
  biology.

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Different alleles of Hb
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Microsatellites and VNTRs as DNA Markers

• Analysis of microsatellites ( short tandem
  repeats or STRs, 2-4 bases repeat), and VNTRs
  (Variable number of tandem repeats, 5- 10s 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 STRs and VNTRs
  than at most sequences in the DNA.

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Microsatellites and VNTRs as DNA Markers
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Analysis of Microsatellites and VNTRs

• 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
• 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.

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Applications

• 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.

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DNA Typing in Paternity Testing

• Comparison of VNTRs can definitely exclude an
  individual from being the parent of a child
  (neither allele the child has is present in the
  alleged father).

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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 VNTRs) 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 VNTRs 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.

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Finding a Gene Chromosome Walking

• 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.

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Isolation of Human Genes

• Positional cloning Isolation of a gene
  associated with a genetic disease on the basis of
  its approximate chromosomal position.

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Cystic Fibrosis Gene

• Cystic fibrosis disease is a common lethal
  disease inherited as an autosomal recessive
  manner.
• 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.

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RFLP markers linked to the CF gene (linkage
studies)

• Screen many individuals in CF pedigrees with a
  large number of RFLPs.
• Use Southern blot analysis and hybridize with
  probes to identify different forms.
• Establish a linkage between the markers and the
  occurrence of the disease.

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Which chromosome?

• Use in situ hybridization, where chromosomes are
  spread on a microscope slide, and hybridized with
  a labeled probe, results are analyzed by
  autoradiography.
• A 3H-labeled RFLP probe showed that CF gene is
  located on chromosome 7.

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Which chromosomal region?

• 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.
• 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.

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Cloning the CF between markers

• Chromosome walking technique is used to walk
  across the chromosome between the markers.
• 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.

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• Chromosome walking uses large cloned DNA
  fragments which overlap.
• Clones are isolated from a library based on
  hybridization to the end of the previous clone.

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Problems

• 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.

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Identifying the CF gene in the cloned DNA

• Use cloned DNA as probes to hybridize with other
  species DNA.
• 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.

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Identifying the CF gene in the cloned DNA

• 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.

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Positional cloning

• 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.

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Chromosome walking

• 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.

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What is the difference between a pseudogene and a
gene.

• 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.

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During positional cloning, four candidate genes
are identified.

• What would be the most convincing evidence?
• 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.

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Suppose DNA typing is used in a paternity case.

• How do exclusion results differ from inclusion
  results?
• 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/activitie
  s/blackett/introduction.html
• ANSWER THE ACTIVITY QUESTIONS
• http//www.biology.arizona.edu/human_bio/activitie
  s/blackett2/overview.html
• PERFORM THE ACTIVITES AND ANSWER QUESTIONS
• Pedigree Collect data Paternity testing
  Missing person RCMP freq. calc.