PCR and FISH

Question 1

What is polymerase chain reaction (PCR)

Answer 1

method to amplify a selected DNA fragment

Question 2

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

Answer 2

need to know sequence of both ends of DNA fragment

Question 3

whats the limitation of PCR

Answer 3

cant multiply fragments over 5000 base pairs

Question 4

What is needed for PCR

Answer 4

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

Question 5

What are the steps of PCR

Answer 5

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^)

Question 6

How can you view the PCR result

Answer 6

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

Question 7

process of gel electrophoresis

Answer 7

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

Question 8

why electrophoresis in gel not liquid

Answer 8

provides more resistance

prevents mixing of molecules

Question 9

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

Answer 9

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

Question 10

What is real time PCR (Quantitative PCR)

Answer 10

Faster than normal- only 15 hrs

relies on fluorescently labelled nucleotides

You can measure intensity of fluorescence

So dont have to use electrophoresis step

Question 11

What is reverse transcription PCR (RT-PCR)

Answer 11

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

Question 12

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

Answer 12

oligo-DT (thymine)

complementary to poly A tail of mRNA

Question 13

What is FISH

Answer 13

Fluorescent in situ hybridization

fluorescent labelled probe with specific pairs with complementary bases

chromosomes can be in interphase or metaphase

Question 14

What is the application if FISH

Answer 14

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.

Question 15

What are the steps of FISH

Answer 15

• 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

Question 16

What is CGH (comparative genomic hybridization)

Answer 16

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.

Question 17

how many centromeres in metaphase chromosomes

Answer 17

2

Question 18

what do centromeres do

Answer 18

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

Question 19

What is kinetocore

Answer 19

helps spindle attach to centromere

Question 20

What are telomeres and what do they do

Answer 20

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

Question 21

Do bacteria have telomeres

Answer 21

no because their DNA is circular

Question 22

What do telomerase do

Answer 22

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

Question 23

How is FISH used to detect aneuploidy

Answer 23

FISH in early human blastomeres

uses locus specific probes for the chromosomes eg 21 or 18

Question 24

FISH used for translocations associated with cancer

Answer 24

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

Question 25

What is multicoloiur FISH

Answer 25

FISH of a 4 cell embryo

nuclei hybridized with many probes