Molecular Diagnostics

Question 1

Southern Blot

Answer 1

DNA

Question 2

Northern Blot

Answer 2

RNA

Question 3

Western Blot

Answer 3

Protein

Question 4

RFLPs

Answer 4

Restriction Fragment Length Polymorphisms

Question 5

Restriction enzymes

Answer 5

Restriction ENDOnucleases, cut from inside DNA

Leave sticky ends, bacterial protection from bacteriophage

Question 6

Common Restriction Enzymes

Answer 6

EcoR1, BamH1, HindIII

Question 7

Gel Electrophoresis

Answer 7

Organizes DNA strands by size of fragments

Question 8

Sticky ends can be joined together

Answer 8

Can combine DNA from different organisms into plasmids

Question 9

PCR

Answer 9

Greatly accelerates construction of human genomic DNA library, polymerase chain reaction

Question 10

PCR Mechanism

Answer 10

1. Apply region of DNA to be amplified
2. Separate the DNA strands and anneal primers
3. DNA synthesis
4. Repeat

**Need to add excess nucleotides, DNA polymerase, Taq

Question 11

Thermus aquaticus

Answer 11

Taq: heat stable DNA polymerase

Question 12

Thermal cycler

Answer 12

Able to denature, anneal and elongate automatically, PCR machine

Question 13

Genomic PCR and RT-PCR

Answer 13

1. Genomic PCR: amplify DNA
2. RT-PCR: reverse transcriptase RNA to cDNA

qRT-PCR: quantitative, how much RNA is there?

Question 14

DNA travels and grows from 5’ to 3’ direction

Answer 14

C-G (3hb) and A-T (2hb)

Question 15

Halt DNA synthesis (for sequencing)

Answer 15

Take out OH, make dideoxyribonucleoside (ddATP, ddTTP, ddCTP, ddGTP)

Prevents strand extension at 3’

Question 16

Fluorochrome

Answer 16

Nucleotide marker, allows for visualization of bands

Question 17

Southern Blot in detail

Answer 17

1. Run DNA on gel
2. Denature – transfer to nitrocellulose or nylon membrane
3. Can hybridize with radioactive probe to visualize
4. Visualize on X-ray

Question 18

Northern Blot and Western Blot

Answer 18

1. Northern Blot: RNA

2. Western Blot: Protein, probe with 1’ and 2’ antibody

Question 19

Microarrays

Answer 19

1. Used to make a DNA profile of a patient, use gene chips
2. Normal DNA tagged red
3. Tumor DNA tagged green
4. Equally normal/tumor = yellow

Can use for diagnostics and to determine treatment

Question 20

Progression of tumor is correlated with change in gene expression

Answer 20

Can be visualized using microarrays

Question 21

If tumor genes are being highly expressed

Answer 21

Then can go with aggressive treatment, if not then no