Molecular Diagnostics

Question 1

Describe the process of hybridization. Why is this useful?

Answer 1

Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form DNA-DNA hybrid or DNA-RNA hybrid

It is useful in detecting and quantifying DNA and RNA

Question 2

What is Southern blotting?

Answer 2

Both the probe and target nucleic acid are DNA

Determines which restriction fragments are associated with a gene

Question 3

What is Northern blotting?

Answer 3

Probe is single stranded DNA and target is mRNA

Measures the size and quantities or mRNA molecular

Question 4

What is Western blotting?

Answer 4

Measures the amount or protein or antibody

Question 5

What is Eastern blotting?

Answer 5

Detects post-translational modifications on proteins

Question 6

Describe the process of Polymerase Chain Reaction.

Answer 6

Double stranded DNA is obtained from the patient/pathogen

DNA is denatured to form ssDNA

Primers flank each end of DNA in 3’-5’ direction

Annealing

Add dNTPs- all 4

Taq Polymerase- synthesizes a copy of DNA by extending the primers on both ends. DNA doubles in each cycle.

Advantage is that a very small amount of DNA is needed to amplify.

Question 7

What is different about quantitative PCR?

Answer 7

In addition to primers, this technique includes a probe which fluoresces only in the presence of the PCR product

Question 8

Describe the process of Restriction Fragment Length Polymorphism (RFLP).

What 3 things is this helpful with

Answer 8

Individuals have very few genomes that differ from each other. By targeting these unique differences with restriction enzymes you are able to differentiate between genomes. Uses DNA finger printing to determine this.

This is useful in:
Forensic analysis
Paternity testing
Disease Detection

Question 9

Describe the process of Variable Number of Tandem Repeats (VNTR). What is this useful for?

Answer 9

Short tandem repeats occur in the genome but varies in individuals. Once these short repeats are identified PCR allows them to be amplified.

Useful in identifying inherited diseases and how severe they are. Ex. Huntington Dz

Question 10

What 5 kinds of drugs are found using recombination proteins? What kind of DNA is utilized to form recombinant proteins?

Answer 10

Insulin
Growth Hormones
Erythropoietin
Clotting factors
Vaccines

cDNA is incorporated into Plasmid Vectors to form recombinant proteins

Question 11

When forming Insulin, different drug companies change a single amino acid to make a different they can sell to control blood sugar. What are the 2 fast acting and a single slow acting form of insulin?

Answer 11

Fast acting:
Lispro
Regular Insulin

Slow Acting:
Glargine

Question 12

What does ELISA test for?

Answer 12

Tests for the levels of specific antigen or antibody concentrations. When an antigen or antibody is present there is a color change. More color change=more Ag

If looking for an Ab, start with an Ag
If looking for an Ag, start with an Ab

Question 13

What is the difference between Indirect ELISA and Sandwich ELISA?

Answer 13

Indirect ELISA- measures the amount of antibody in a sample.

Sandwich ELISA- measures the amount of an antigen in a sample. Sandwiches the Ag between the Abs.

Question 14

What is Indirect ELISA used to detect? What test do you confirm with?

Answer 14

Diagnosing HIV

Confirm with Western Blotting

Question 15

What 2 reasons would we utilize Sandwich ELISA?

Answer 15

MI detection

Pregnancy Test

Question 16

How is Sandwich ELISA used in a pregnancy test? What are the 3 sites on a pregnancy stick and what occurs at each site?

Answer 16

Reaction Site:
Free hCG Abs, which bind to hCG in the urine. This complex moves to the test site

Test Site:
Immobilized hCG Ab hCG/ab complex from the reaction site binds to immobilized hCG Ab. Sandwich is complete and gives dye its color.

Control Site:
Non-specific Ab is immobilized
Dye gives color regardless of positive or negative test of hCG. Confirms that this test is working.