MCQ 2 Flashcards
Southern blotting
A method to detect single stranded DNA that has been transferred to nylon paper by using a probe that binds DNA
Northern blotting
Hybridisation technique where a DNA probe binds to an RNA target molecule
Steps of Southern blotting
Target DNA cut with restriction enzyme
Fragments separated by gel electrophoresis
dsDNA melted by soaking gel in alkaline denaturing solution
DNA fragments transferred to nylon membrane
Membrane dipped in solution of labelled DNA probes molecules
What’s an example of a alkaline denaturing solution?
Sodium hydroxide
How do the DNA probe molecules work in Southern blotting?
They only bind to DNA fragments with similar sequences
Makes area radioactive
A black spot will appear where the hybrid molecule is if a piece of photographic film is placed on top
What makes the primers needed for PCR?
Primase
Why is SYBRR green a useful reagent for real time PCR?
It flouresces only when bound to dsDNA
What are the mains things needed in a PCR reaction mixture?
DNA polymerase
Primers
Deoxyribosenucleoside triphosphates
What type of template is needed for RT-PCR
RNA template
What type of template is needed for inverse PCR?
A circular template
Which variant of PCR may be used to selectively amplify mRNAs from a sample of total cellular RNA?
Differential display PCR
What are the steps and temperatures of DNA in order?
Strands melt @ 90oC
Primers anneal @ 50-60oC
DNA synthesis @70oC
What is responsible for DNA synthesis in PCR?
Taq polymerase
Inverse PCR
Method for using PCR to amplify unknown sequences by circularising the template molecule
When do you use inverse PCR?
When you know a bit of a DNA sequence in the middle but not the bits either side
Degenerate primer
A primer with several alternative bases at certain positions
Long PCR
PCR reaction used specifically to amplify longer target sequences than standard PCR
Hot start PCR
Taq polymerase is kept inactive at low temperatures by antibodies or blocking proteins to make sure template DNA is fully denatured
Unique steps of inverse PCR
Restriction enzymes cut DNA sequence, but not known area
Fragment ligated so it circularises
Primers used that face outwards from known sequence
PCR run on circular DNA
What does inverse PCR result in?
Strands of DNA with known sequence at the ends
Randomly amplified polymorphic DNA (RAPD)
Method for testing genetic relatedness using PCR to amplify arbitrarily chosen sequences
Reverse transcriptase PCR
Allows genes to be amplified and cloned intron free
Uses mRNA and reverse transcriptase to make copies (cDNA)
Differential display PCR
Variant of RT-PCR that specifically amplifies mRNA from eukaryotic cells using oligo(dT) primers
Oligo(dT) primer
Primer used on 3’ end of cDNA in differential display PCR
Rapid amplification of cDNA ends (RACE)
RT-PCR based technique
Uses internal primers to speed to up copying
Can work 3’ to 5’
Real time fluorescent PCR
Uses DNA binding probes that fluoresce more or only when bound to dsDNA
As more dsDNA is produced, fluorescence increases as more probe binds
Multiplex PCR
Real time PCR reaction
Uses multiple fluorescent dyes on different probes
Assesses amplification of more than one target sequences
Long interspersed element
Makes up majority of moderately repetitive DNA
Long sequences of DNA
Found in multiple copies
Short interspersed element
Short repetitive sequences of DNA that are highly repetitive
Satellite DNA
Highly repetitive DNA of eukaryotic cells
Found as long clusters of tandem repeats
Permanently coiled tightly into heterochromatin
Tandem repeats
Repeated sequences of DNA/RNA that lie next to each other
How many base pairs in a microsatellite?
~13
How mant base pairs in a minsatellite?
~25
Variable number tandem repeats (VNTR)
Cluster of tandemly-repeated sequences in DNA
Numbers of repeats differs from one individual to another
What is used in DNA fingerprinting?
Variable number tandem repeats (VNTR)
