Lec 68: Part Two: Clinical Genetic Techniques

Question 1

Function of Cytogenetics

Answer 1

Using fresh samples, you can determine numerical and structural abnormalities

Question 2

General Stepwise Function for Cytogenetic Process

Answer 2

1. Arrest Cells at metaphase using drugs
2. Aspirate cells to release Chromosomes
3. Use special stains to ID bands

This is the general idea, all of them deviate from this in their own ways

Question 3

How does DNA sequencing work?

Answer 3

Nucleic acid hybridization - Use a complementary fluorescent base pairing to a specific sequence

Question 4

FISH

Answer 4

Fluorescent In Situ Hybridization - Uses fluorescently labeled ssRNA that will bind to any and all present complimentary strands. This is great if you have limited tissue and it doesn’t require arresting the cells

Question 5

CGH Array

Answer 5

Label the patient’s DNA green, the control standard normal DNA red, put them in all kinds of wells with all of the genomic nucleotides. If you get anything but a mix of green and red in each well, then you are good. If you get more red or more green, then you have more or less patient DNA = abnormal.

Question 6

SNP Array

Answer 6

Single Nucleotide Polymorphism Array - Can detect parental disomy like in Angelman’s and can tell you copy number through signal intensity. Can also tell you Zygosity based on present allele.

Question 7

Sanger Sequencing

Answer 7

The GOLD STANDARD - Needs a lot of DNA, but by adding in fluorescent ddNTPs, ddATPs, etc, you can deduce the nucleotide sequence. This is very labor intensive

Question 8

What is the new age version of Sanger Sequencing?

Answer 8

Pyrosequencing

Question 9

Pyrosequencing

Answer 9

Uses methodology of Sanger Sequencing. Uses Luciferase and adds one nucleotide at a time. Spikes on the computer tell you where in the sequence that nucleotide is. This is perfect for unknown mutations

Question 10

Maternal Free Serum DNA testing

Answer 10

Non invasive. 10% of mom’s serum has fetal blood, so you can piece this together to determine fetal genome. Be warned, only use this as a counting mechanism, not a strict sequencer.

Question 11

What is q on a chromosome mean?

Answer 11

Long arm

Question 12

What is p on a chromosome?

Answer 12

Short arm (think petite)

Question 13

What is the nomenclature for determining where you are on a chromosome?

Answer 13

, arm, region, band, sub band

Ex. 15q13.2 would be the 15th chromosome, the long arm, region 1, band 3 sub band 2.

Question 14

PCR

Answer 14

Polymerase Chain Reaction - Selectively Amplifies Target DNA sequences up to a million fold!

Question 15

PCR Materials

Answer 15

Template DNA
2 primers at least
Taq DNA polymerase
4 deoxynucleotide triphosphates (lots of each one)
Mg++ Buffer solution

Question 16

PCR Recipe

Answer 16

1. Denature DNA at high temperature to separate strands
2. Bring temperature back down = annealing and hybridizing of the DNA
3. Repeat A LOT
4. Run on a gel and stain with Ethydium Bromide. View under UV light

Question 17

Real Time PCR

Answer 17

Same as old method, just updated. Tells you whats happening via a reporter signal on ddNTPs so you don’t need a gel, computer does it

Question 18

What is “Next Generation Sequencing?”

Answer 18

Billions of sequences at once allowing breadth while doing same genes over and over for depth. Tells you everything you need to know.

Question 19

How do restriction endonucleases hep us?

Answer 19

Use them with PCR product. They are mean’t to cut certain sequences. If you have mutations, particularly point mutations, you won’t cut certain pieces. Compare this mess with the standard to see if you have more or less of the pieces.