Lec 68: Part Two: Clinical Genetic Techniques Flashcards Preview

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Flashcards in Lec 68: Part Two: Clinical Genetic Techniques Deck (19):

Function of Cytogenetics

Using fresh samples, you can determine numerical and structural abnormalities


General Stepwise Function for Cytogenetic Process

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


How does DNA sequencing work?

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



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


CGH Array

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.


SNP Array

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.


Sanger Sequencing

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


What is the new age version of Sanger Sequencing?




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


Maternal Free Serum DNA testing

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.


What is q on a chromosome mean?

Long arm


What is p on a chromosome?

Short arm (think petite)


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

#, arm, region, band, sub band

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



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


PCR Materials

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


PCR Recipe

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


Real Time PCR

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


What is "Next Generation Sequencing?"

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


How do restriction endonucleases hep us?

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.

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