Introduction, Evolution and Genomes-10 Flashcards
PCR and sequencing
How are mechanisms of DNA replication characterised?
At a molecular level.
What is DNA sequencing?
Order of nucleotides in piece of DNA.
What did Walter Gilbert discover?
Partial chemical degradation of radiolabelled DNA.
Biology labs, Harvard.
What did Frederick Sanger discover?
Enzyme-mediated incorporation of dideocynucleotides into newly-replicated DNA.
MRC labs, Cambridge, 1970s.
What does Sanger sequencing of dideoxynucleotides rely on?
On the incorporation of dideoxynucleotides into newly replicated DNA.
What does dideoxynucleotide do?
Is a terminator.
Need the 3’ hydroxy; from the previous nucleotide.
When it is incorporated into the DNA, it becomes a terminator.
Nucleotide that lacks the 3’, can no longer make an ester bond with a new nucleotide.
What is the principle of Sanger’s dideoxy sequencing method?
Add: a single stranded template DNA, a printer complementary to part of this template, DNA polymerase, a pool of normal deoxynucleotides (dATP, aTTP, DGTP and dCTP) and a small portion of radioactive-labelled ddATP.
Some newly synthesised molecules will get a ddATP in each place that there is a T in the template DNA.
The result is a nested set of new DNA molecules each of which ends in ddA.
How is Sanger’s dideoxy sequencing method done?
Take tube, template and specific primer.
Divide this into 4 tubes, add a small amount of ddG, ddA, ddT and ddC into each one.
Different products form for each ddNTP and they all terminate.
Can determine them by size, using gel electrophoresis- small fragments move further than larger fragments, top of the gel corresponds to the 3’ end, the results are the order of bases in the DNA.
What is automated dideoxy sequencing?
Template + primer + DNA Pol + all dNTPs + all fluorescent ddNTPs in one tube.
The dye present in each synthesised fragment corresponds to the dye attached to the dideoxynucleotide that terminated the synthesis of that particular fragment.
Pass nested products through an electrophoretic system and reach with lasers.
It is cheap and reliable.
What is PCR? Who invented it? When?
Is a ‘copying machine’ for DNA- a tool to amplify a specific DNA sequence.
Kary Mullis. 1983. Resulting in his Nobel Prize in Chemistry.
Give a brief history of DNA?
1953- Watson and Crick: DNA model.
1957- Kornberg: first DNA polymerase.
1960s- Har Gobind Khorana: started deciphering the genetic code.
1969- Thomas Brock: isolated bacterium, Thermus aquaticus, from a hot spring in Yellowstone National Park.
1971- Khorana’s group suggested that a template-primer-polymerase system could be used for copying DNA, and using two primers should lead to replication of a specific fragment of DNA (but did not show results).
1976- Taq polymerase was isolated from T. aquaticus.
1977- Sanger: ddDNA sequencing.
What are the components of PCR?
Template dsDNA with a target area, and DNA sequence knowledge.
Two specific oligodeoxynucleotide primers (typically 20-24 mers) which can hybridise at each end of the target sequence.
dNTPs.
Buffer and MgCl2.
Taq polymerase.
Why is Taq polymerase useful?
Thermus aquaticus, a Gram-negative rod, was isolated form this spring, from the outflow (70 degrees).
T. aquaticus thrives at ~70 degrees but can survive at temperatures between 50 to 80 degrees.
PCR is possible because of the thermal stability of Taq DNA polymerase.
What is the PCR method?
Amplification of the target DNA is achieved by repeated cycles of:
-heating (94) to denature the target DNA. Taq polymerase is not denatured by this.
-hybridisation of primers, one to each strand (45-65).
-extension of the primers by Taq DNa polymerase (72).
Typically ~30 cycles are performed. Care over temperature control is essnetial.
PCR machines are programmable for the use with many different cycling parameters and very rapidly change the temperature between the various stages of the PCR process.
What happens in Cycle 1 of PCR?
Heat to 94, 1-2 min, to separate the strands.
Cool to ~50 to allow ‘primers’ to bind.
Heat to 7, 1-2 min, to allow Taq polymerase to ‘extend’ the primers.
The sequence is specified by the target and primers used.
Start: one ds copy of what you wish to amplify. End: two copies of what you want.
What is exponential amplification?
When you plot it onto a linear scale it looks like nothing happens at first.
The cycle is repeated about 30 times, automated. 30 cycles = >1000 million copies.
Why is agarose gel used?
To visualise the PCR reactions.
What is agarose gel electrophoresis?
The reactions are loaded into the wells of a slab gel made of agarose.
An electric current is applied.
DNA is negatively charged, so it migrates to the positive terminal.
The gel contains a dye that binds DNA, so the DNA will fluoresce under the UV light.
Large fragments migrate slowly, small fragments migrate quickly.
How can DNA be examined by PCR reactions?
From tiny amounts of DNA we can amplify large visible amounts.
Starting material: undetectable. After 30 cycles: clearly visible.
What is sensitivity and specificity?
Sensitivity- one target molecule can be amplified to >10^9 in just a few hours, because a product formed in one cycle becomes a template for the next.
Specificity- is provided by the primers, they are complementary to opposite strands with therr 3’ ends pointing towards each other.
What are the applications of PCR?
Amplify tiny amounts of DNA to obtain enough for analysis by almost any of the unusual molecular biological techniques.
Analyse small samples for medical genetic purposes.
Analyse tiny explants of tissue (e.g. chorionic villus samples) for pre-natal screening.
Amplify archaeological and ancient DNA sources, use PCR to alter a DNA sequence and forensic applications.
How can specific mutations be made?
Mutations can be introduced into amplified DNA by engineering one or more mismatches into a primer close to or at its 5’ end.
Target DNA duplex, denaturation and primer binding, final mutated product.
What is the sequencing of archaic hominin genomes?
Nuclear DNA was amplified from fossil Neanderthal bones from the Vindija site and compared with DNA from modern humans.
Conclusions: Europeans and Asians have ~4.5% (now revised as 2%) of their genes derived from Neanderthals. Gene flow can be detected from Neanderthals into modern humans, but not the reverse and no mtDNA gene flow has been detected. This suggests male Neanderthal and human couplings. DNA amplification and sequences are re-writing our origins.
How can DNA profiling be used in forensic science?
PCR amplifies target DNA, so very small samples can be processed.
- Sir Alec Jefferys. VNTRs/STRs can be used to identify people.
Using multiple STRs increases discrimination.
What is a VNTR?
Variable Tandem Repeat.
Run of short, repeated nucleotide sequences (typically 3-5 bp).
Can be found on many chromosomes and after show variations in length between individuals.
Why will individuals usually inherit a different variant of each STR locus from their mother and father?
STR acts as an inherited allele.
The number of repeats is highly variable in the population.
Thus, STR can be used for personal or parental identification.
What is CODIS?
Combined DNA Index System, operated by the FBI.
Typically a multiplex PCR is performed (multiple reactions in one tube) using specific primers for 13 different STRs on different chromosomes.
Also a European standard set. US and EU sets can identify every person in the world, as long as X/Y status is included.
