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.
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
General screening and genetic testing are different
(mammograms vs. testing for specific mutations in
Prenatal diagnosis: is the fetus at risk?
(amniocentesis or chorionic villus
Newborns can be tested for PKU, sickle
cell anemia, Tay-Sachs.
Methods of Genetic Testing
Restriction Fragment Length
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.
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
Microsatellites and VNTRs as
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.
Analysis of Microsatellites and
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
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
DNA typing: paternity testing (also useful in
population studies, in animal breeding etc.)
and in forensic analysis.
DNA Typing in
• 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
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
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
Isolation of Human Genes
Positional cloning: Isolation of a gene
associated with a genetic disease on
the basis of its approximate
Cystic Fibrosis Gene
Cystic fibrosis disease is a common lethal
disease inherited as an autosomal recessive
Identify RFLP markers linked to the CF gene.
Identify the chromosome on which the CF gene is
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)
Screen many individuals in CF
pedigrees with a large number of
Use Southern blot analysis and hybridize
with probes to identify different forms.
Establish a linkage between the markers
and the occurrence of the disease.
Use in situ hybridization, where
chromosomes are spread on a
microscope slide, and hybridized with a
labeled probe, results are analyzed by
A 3H-labeled RFLP probe showed that CF
gene is located on chromosome 7.
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.