PCR and FISH Flashcards

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1
Q

What is polymerase chain reaction (PCR)

A

method to amplify a selected DNA fragment

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2
Q

What do we need to know in order to run a PCR

A

need to know sequence of both ends of DNA fragment

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3
Q

whats the limitation of PCR

A

cant multiply fragments over 5000 base pairs

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4
Q

What is needed for PCR

A

DNA polymerase-forms phosphodiester bonds+ proof reading ability- can cut out nucleotide
from thermal stable bacteria

primers- short oligonucleotides that are complementary to the sequence . starts off synthesis- polymerase needs a nucleotide to spot

thermal cycler

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5
Q

What are the steps of PCR

A

denaturation- 90’c to break hydrogen bonds between strands

Annealing - variable but 55’c? complementary binding of primers to template

Extension-70’c DNA polymerase binds to 3’ of primer

after whole fragment made, cycle repeated
* fragment of interest amplified exponentially ( 2^)

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6
Q

How can you view the PCR result

A

gel electrophoresis
* requires 2 hrs of electricity
* stain gel with fluorochrome
* long process 2 days

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7
Q

process of gel electrophoresis

A

DNA has negative charge due to phosphates so migrates to anode (+)

Shorter fragments move faster as less resistance

DNA stained with fluorochrome

UV lets us see amplified fragments as bands

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8
Q

why electrophoresis in gel not liquid

A

provides more resistance

prevents mixing of molecules

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9
Q

What do you have to do different if you do protein electrophoresis

A

you have to artificially charge them as different amino acids have different charges

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10
Q

What is real time PCR (Quantitative PCR)

A

Faster than normal- only 15 hrs

relies on fluorescently labelled nucleotides

You can measure intensity of fluorescence

So dont have to use electrophoresis step

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11
Q

What is reverse transcription PCR (RT-PCR)

A

relies on RNA template NOT DNA

the reverse transcriptase is used to make cDNA (single stranded)

cDNA binds with primers and DNA polymerase steps in

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12
Q

what is the first primer of RT-PCR and what is it complementary to

A

oligo-DT (thymine)

complementary to poly A tail of mRNA

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13
Q

What is FISH

A

Fluorescent in situ hybridization

fluorescent labelled probe with specific pairs with complementary bases

chromosomes can be in interphase or metaphase

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14
Q

What is the application if FISH

A

Used for finding specific features in DNA for use in genetic
counselling, medicine, and species identification.

identifies the location of a particular sequence on metaphase and interphase chromosomes-played imp role in mapping genes on chromosomes

  • analyze chromosomes from tumours
  • testing of numerical and structural chromosome aberrations in human.
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15
Q

What are the steps of FISH

A
  • cells fixed on slide. fluorescently labelled probe added
  • specimen and strand treated with heat to become single strand
  • if probe finds complementary sequence then they hybridize
  • non hybridized probe washes away
  • nucleus stained with fluorescent dye as well. View under fluorescent microscopy
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16
Q

What is CGH (comparative genomic hybridization)

A

Compares 2 DNA sequences of the same person
- one strand normal one strand with deletions or duplications

CGH allows observation of very small deletions and duplications, not detectable by banding and FISH.

Tumor DNA and normal (control) DNA are labelled with different fluorochromes and hybridized to normal diploid metaphase chromosomes on a microscope slide.

17
Q

how many centromeres in metaphase chromosomes

A

2

18
Q

what do centromeres do

A

In interphase they help in the organization of loops of chromosomes

In metaphase, they help keep sister chromatids together
* rings of cohesin open
* ring in centre holds it together
* ring destroyed in anaphase which split the chromatids

19
Q

What is kinetocore

A

helps spindle attach to centromere

20
Q

What are telomeres and what do they do

A

They are on the ends of chromosomes

Protect chromosomal ends , they are repetitive, non coding sequences

  • they become shorter with each division (as we age) - only the repeats gets lost, not the actual coding genes
  • when telomere is so short, no more cell division occurs as they cant cut the real genes therefore apoptosis
21
Q

Do bacteria have telomeres

A

no because their DNA is circular

22
Q

What do telomerase do

A

They add extra telomeres to elongate the ends

  • good tumour marker- uncontrollable= cancer
  • It attaches to maternal strand in order to elongate the daughter strand
  • uses reverse transcription to elongate telomeres
23
Q

How is FISH used to detect aneuploidy

A

FISH in early human blastomeres

uses locus specific probes for the chromosomes eg 21 or 18

24
Q

FISH used for translocations associated with cancer

A

2 genes involved in translocation are labelled in different colours
normal cells have 2 signals for each colour

If translocation occurred, part of one chromosome goes to other and vice versa and then fuse, both colours together

25
Q

What is multicoloiur FISH

A

FISH of a 4 cell embryo

nuclei hybridized with many probes