Molecular Diagnostics

Question 1

Southern blotting

Answer 1

Both probe and target nucleic acid are DNA

Determine which restriction fragments are associated with a gene

Question 2

Northern blotting

Answer 2

Probe is ssDNA and target is mRNA
Measure size/quantities of mRNA molecules (questions about gene expression)
SNOW
DROP

Question 3

Western blotting

Answer 3

Targets proteins, can measure amount of protein or antibody present
SDS-PAGE separates protein using electrical field
Smaller proteins move faster than larger

Question 4

Eastern blotting

Answer 4

Detects post translation modifications on proteins

Question 5

PCR

Answer 5

Denature DNA, add primers that complement sequences that flank each end of DNA
Allow them to anneal, then add all for dNTPs
Taq polymerase extends primers on both ends

Question 6

PCR advantage/disadvantage

Answer 6

Very small amount of DNA needed to make large amount

Need to know sequence of flanking DNA for primer, error prone, amplification of contaminated DNA

Question 7

PCR purpose

Answer 7

Earlier detection of microorganisms (i.e. HIV)

Detection of genetic mutations- cystic fibrosis, thalassemia

Question 8

Quantitative PCR - qPCR

Answer 8

Used to detect levels of an infections agent, determine levels of gene expression
Quantifies copy number of a specific gene

Question 9

Restriction fragment length polymorphism RFLP

Answer 9

Individuals genomes differ by 1 in every 1000 base pairs
Some of these differences occur in the recognition sequences for restriction enzymes
Used for forensic analysis, paternity testing, disease detection

Question 10

VNTR

Answer 10

People have differences in pattern of short tandem repeats
Can be detected by PCR or flanking restriction sites
Useful in identification and severity of inherited diseases such as huntingtons and fragile x
Used in newborn screening

Question 11

Human insulin vs Lispro and insulin aspart

Answer 11

Human has proline at position 28 and lysine at 29
Lispro switches those two AAs
Insulin aspart replaces proline with aspartate
They are faster acting and more readily absorbed

Question 12

ELISA

Answer 12

Tests for levels of specific antigen or antibody concentrations
When antigen or Ab bind target, a color change occurs

Question 13

Indirect ELISA

Answer 13

Measures the amount of antibody in a sample (well is coated with antigen, Ab binds, then enzyme linked antibody is added)
Used to test for HIV (must be confirmed using western blotting)

Question 14

Sandwich ELISA

Answer 14

Measures the amount of antigen in a sample (well Is coated with antibody, Ag binds, then enzyme coated Ab is added)
Pregnancy test, recent myocardial infarction test