9 - Genetic Testing

Classic genotype phenotype concordance vs genetic heterogeneity vs phenotypic pleotropy

1. Classic genotype phenotype concordance: One gene causes one disease

2. Genetic heterogeneity: One disease caused by multiple genes

3. Phenotypic pleiotropy: One gene causes multiple diseases

What is called when we do a test on someone who already has signs/symptoms of a disease and we want to confirm/rule out causes?

• Diagnostic testing

What is carrier screening, prenatal testing, preimplantation genetic testing?

1. Carrier screening: Testing to see if we are a carrier of a recessive disease causing gene

2. Prenatal testing: Testing fetus during pregnancy to see if they have any abnormalities

3. Preimplantation genetic testing: Testing embryos before implantation(For couples w 2 or more miscarriages due to genetic abnormalities)

What is predictive testing?

A test to see if we will develop a condition in the future

• Presymptomatic: Test for a disease with high penetrance

• Predispositional: Test for a disease with low penetrance

What substance should we not use to collect fresh whole blood and why?

Heparin; interferes with PCR

Sanger Sequencing

• What is it

• Pros and cons

• A DNA test that utilizes ddNTPs, or nucleotides that end the polymerase ability to continue if they are added to the DNA chain. There will be millions of copies of our target sequence to be replicated, and a pool of dNTPs and ddNTPs, and on average, there should be copies where the DNA polymerase added a ddNTP at each position, so we then separate them by size and see what each base is in each spot

• Accurate and reliable but not sensitive to low frequency variations and slow

PCR

• What is it

• Pros and cons

• A DNA test that multiplies one copy of DNA until there are millions, and these copies can be used in Sanger Sequencing, infectious disease identification, forensics

• Sensitive and cheap but we can’t actually look at the sequence in detail itself unless we do a next step

MLPA

• What is it

• A test to see if any copy number variants(extra copies of/deletions of a gene) are present; it’s done by adding 2 probes that are supposed to be next to each other on a gene, and so they should find each other and bind, and then we do PCR to amplify these probes. We then look at how many copies were produced, if it’s proportional to a patient having 2 genes like normal, less(a deletion), or more(duplication)

Linkage Analysis

• What is it?

• A genetic testing process used to roughly identify where a disease causing gene is. Because areas close to each other on chromosomes usually travel together, we can look at many generations of a family members to see if the sick individuals have any shared sequences, and we can tell that that area might be the area that codes for the disease to happen

Inverse PCR

• What is it?

• A method where we cut out a region of DNA including a known region, and we turn the DNA into a circle, and use the primer to go OUTWARD and replicate that whole circle, and then amplify it. Now, we have the gene regions slightly up and downstream of the known region

NGS

• What is it?

• A DNA test that shreds the sample into millions of small fragments, amplifies each into a cluster (like PCR), then reads all clusters simultaneously by adding one fluorescently-tagged base at a time and imaging the whole surface after each cycle. The many short reads are then computationally overlapped and reassembled into the full sequence.