239b molecular dx

Question 1

what is the point of using molecular dx?

Answer 1

to detect genetic variation (sequence, CNV, expression, epigenetic)

Question 2

methods to detect specific genetic variation (prior knowledge of targeted sequence)

Answer 2

PCR (uses DNA)

RT-PCR (reverse transcriptase - targets RNA with primers on exons) –> make cDNA from RNA and then dsDNA –> continue as with PCR

gel electrophoresis with products of PCR

real-time PCR - fluorescence molecular beacon that lights up more when part of dsDNA

Question 3

CT value with PCR

Answer 3

standard curves (from known samples) allow determination of number of copies of a DNA segment based on how many cycles it takes to be detectable

allows quantitative PCR

Question 4

enzyme linked immunosorbent assay (first aid)

Answer 4

Used to detect the presence of either a specific antigen (direct) or a specific antibody (indirect) in a patient’s blood sample. Patient’s blood sample is probed with either:

Indirect ELISA: uses a test antigen to see if a specific antibody is present in the patient’s blood; a secondary antibody coupled to a color generating enzyme is added to detect the first antibody.

Direct ELISA: uses a test antibody to see if a specific antigen is present in the patient’s blood; a secondary antibody coupled to a color-generating enzyme is added to detect the antigen.

If the target substance is present in the sample, the test solution will have an intense color reaction, indicating a + test result.

Used in many laboratories to determine whether a particular antibody (e.g., anti-HIV) is present in a patient’s blood sample. Both the sensitivity and specificity of ELISA approach 100%, but both false-positive and false-negative results occur.

Question 5

methods to detect CNV from chromosomes to exons

Answer 5

karyotyping of metaphase chromosomes - trisomies, sex chromosome disorders

FISH - fluorescent DNA/RNA probe binds specific gene to localize and visualize anomalies; need large segment deletion/addition

array CGH - detects SNPs and CNVs via binding to sequences on a chip; can’t detect balanced rearrangements (b/c DNA is cut up into tiny pieces)