DNA molecular testing and DNA Typing

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.

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).

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.

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.

RFLPs 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

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

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.

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.

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

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 Positional cloning: Isolation of a

gene associated with a genetic disease on the basis of its approximate chromosomal position.

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.

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.

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.

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.

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.

• 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.