Sequencing and PCR Flashcards
What is DNA hybridisation?
Pulling apart 2 strands of DNA then label particular sequence and put back together to find complementary sequence.
What is agarose?
Seaweed carbohydrates heated in buffer to dissolve.
Forms a gel through which a current can flow.
Polymerised agarose is porous and allows for separation of DNA by charge and size.
How is agarose gel electrophoresis conducted?
Wells are made in the gel where DNA, tracking dye, and glycerol are added near the negatively charged cathode. -ve DNA migrates to positive electrode.
What does the rate at which DNA migrates depend on?
Strength of electric field
Buffer
% of agarose in gel
Size of the DNA (smaller moves faster than large)
How is DNA migration on electrophoresis related to size?
Linear DNA migration is inversely proportional to log10 of molecular size
What kinds of dye are used for gel electrophoresis?
Dyes that bind DNA and fluoresce under UV light to allow visualization such as ethidium bromide and GelRed.
How can we determine the approximate number of bases in a DNA sample?
Using standards and then determine size of sample relative to the standards
What is the substrate for DNA polemerase?
Deoxynucleoside triphosphate
What is required for DNA polymer formation (DNA replication)?
Free 3’-OH
DNA polymerase
dNTP
Template strand
Short double stranded NA
MG2+
What is azidothymidine?
A nucleoside like molecule that doesn’t have an OH (Na+ instead) on 3’ end preventing elongation of NA strand.
What is dideoxyribonucleoside triphosphate?
Molecule with a H instead of OH at 3’ end ending the elongation of nucleotide sequence. This is used for Sanger sequencing
How are nucleotides labelled in modern times?
Using radiolabels or a fluorescent dye
What is PCR?
In vitro amplification of a region of DNA whose sequence is known or which lies between 2 regions of known sequence
How is DNA polymerase in PCR stable at higher temperature?
Due to Taq polymerase created by thermostable bacteria in hot springs of yellowstone national park.
What is required for PCR?
DNA template to copy
Primers to provide 3’OH
Enzyme (Taq DNA polymerase)
dNTPs
Mg2+
Buffer
Thermocycler (cycles between high temperature and low temperature)
Why is a thermocycler necessary for PCR?
To allow the DNA to separate and then it is cooled to allow ligase to bind some primers to the DNA strand and then the strand can replicate at hot temperature.
What are the types of primers needed for PCR?
2 sets: Forward and reverse. One binds to each template strand.
Why are primers not made up of inverted repeats ever?
To prevent formation of hairpins.
What bases make up primers for the most part?
GC is preferred for best annealing. (40 - 60% of bases)
What part of the DNA are primers complementary to?
3’ ends of target DNA. They are synthetically produced.
Taq DNA pol has 5’ - 3’ activity only and does not proofread. Is that an issue for PCR?
No, PCR is a qualitative assessment of a gene so if a few are mutated that isn’t an issue.
What are the 3 stages of PCR?
Denaturation of DNA +/- 95 degrees (templates)
Primer hybridisation/annealing +/- 50 degrees
DNA synthesis (extension) +/- 72 degrees
At what rate are sequences amplified in PCR?
Exponentially.
How are PCR amplification products identified?
Via gel electrophoresis
Sequencing of amplified fragment
Southern blot
What is PCR used for?
Diagnostics
Evolution studies
Detection of drug resistance genes
Forensics (DNA fingerprinting)
