PCR and FISH Flashcards
What is polymerase chain reaction (PCR)
method to amplify a selected DNA fragment
What do we need to know in order to run a PCR
need to know sequence of both ends of DNA fragment
whats the limitation of PCR
cant multiply fragments over 5000 base pairs
What is needed for PCR
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
What are the steps of PCR
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^)
How can you view the PCR result
gel electrophoresis
* requires 2 hrs of electricity
* stain gel with fluorochrome
* long process 2 days
process of gel electrophoresis
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
why electrophoresis in gel not liquid
provides more resistance
prevents mixing of molecules
What do you have to do different if you do protein electrophoresis
you have to artificially charge them as different amino acids have different charges
What is real time PCR (Quantitative PCR)
Faster than normal- only 15 hrs
relies on fluorescently labelled nucleotides
You can measure intensity of fluorescence
So dont have to use electrophoresis step
What is reverse transcription PCR (RT-PCR)
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
what is the first primer of RT-PCR and what is it complementary to
oligo-DT (thymine)
complementary to poly A tail of mRNA
What is FISH
Fluorescent in situ hybridization
fluorescent labelled probe with specific pairs with complementary bases
chromosomes can be in interphase or metaphase
What is the application if FISH
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.
What are the steps of FISH
- 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
What is CGH (comparative genomic hybridization)
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.
how many centromeres in metaphase chromosomes
2
what do centromeres do
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
What is kinetocore
helps spindle attach to centromere
What are telomeres and what do they do
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
Do bacteria have telomeres
no because their DNA is circular
What do telomerase do
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
How is FISH used to detect aneuploidy
FISH in early human blastomeres
uses locus specific probes for the chromosomes eg 21 or 18
FISH used for translocations associated with cancer
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
What is multicoloiur FISH
FISH of a 4 cell embryo
nuclei hybridized with many probes
