Manipulating Genomes Flashcards by Katie Jenman

What is PCR

A method of amplifying dna via artificial replication in vitro (outside of the body)

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Requirements of PCR

-Original DNA sample (less than 10000 bases)
-Free DNA nucleotides
-DNA (taq) polymerase
-Small primer sequences
-Thermocycler (machine that varies temperature precisely)

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Stages of PCR and temperatures at which they occur

-Denaturation (around 95C)
-Annealing (around 55-68C)
-DNA Synthesis (around 72C)

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Describe the denaturation stage

-Happens at around 95C
-Temperature is raised so hydrogen bonds between bases are broken and the original strand becomes two single strands.

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Describe the annealing stage

-Happens at around 55-68C (temperature is dropped a bit so things can bind to the single strands)
-Short primer sequences bind to the 3’ ends of the single strands

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Describe the DNA synthesis stage

-Happens at around 72C (ideal temp for taq polymerase meaning faster reaction)
-Taq polymerase binds to the primers and moves along the strand adding the complementary bases in order to make two new strands

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How to set up for PCR to occur

-Mix DNA sample, nucleotides, taq polymerase and primer sequences in a PCR tube and placed into a thermocycler

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How long does each PCR cycle take

-Around 2 minutes, the amount of DNA grows exponentially

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What are the 4 applications of PCR

-Forensic science applications
-Medical applications
-Infectious disease applications
-Research applications

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What are the forensic science applications of PCR

-DNA profiling, small traces of DNA can be amplified and matched with potential suspects.
-Determining genetic relationships during immigration cases or custody disputes

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What are the medical applications of PCR

-DNA sequencing where mutations may be detected by looking at which bases have changed
-Genetic tests where parents may see if they are carriers of disease
-Tissue typing where the compatibility between tissues from the donor and the recipient can be tested for any chance of rejection (because antigens are proteins coded for in DNA)

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What are the infectious disease applications of PCR

-PCR tests can detect traces of viral DNA in host cells or the bloodstream earlier so antiviral treatments can begin sooner
-Can help monitor spread of infectious diseases by detecting new subtypes of pathogen so scientists can prepare for outbreaks

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What are the research applications of PCR

-Can amplify ancient DNA of extinct organisms to determine evolutionary relationships between species
-Can study gene expression to understand how genes work to develop organisms and keep them alive

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What is electrophoresis

-The separating of DNA fragments according to size (variation of chromatography that moves macromolecules by applying electric current)

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Requirements of gel electrophoresis

-Separated DNA molecules (between 100-25000 base pairs)
-Agarose gel plate
-An electrophoresis tank
-Buffer solution (ion containing solution)
-DNA loading dye
-DNA ladder

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Process of gel electrophoresis

-DNA loading dye is added to PCR tube which contains DNA samples
-This mixture is added to the wells of the agarose gel plate via pipette
-Electric current is passed through and bubbles may start to appear if the DNA is running
-DNA (overall negative charge) will travel towards the anode
-Smaller DNA fragments will move faster towards the anode

What is DNA profiling?

A method used to produce a specific pattern of DNA bands which is unique to an individuals genome

What are VNTRS and what is their use

VNTRS (variable number tandem repeats) are short, repeating sequences of DNA found within non coding regions of DNA.
They determine how long the fragment of DNA is due to their varied number of repeats.

What are the 4 stages of DNA profiling?

Extraction
DNA amplification
DNA digestion
Gel electrophoresis

What is the extraction stage of DNA profiling

The initial stage
Where the DNA is collected from tissues and cells
DNA can be collected using mouth swabs, from remains of blood, hair or skin cells or from bone marrow (helps with ancient DNA)

What is the DNA amplification stage of DNA profiling?

Second stage
Strand of DNA is replicated using the polymerase chain reaction, this means that if one strand is damaged, there are more to work with.

What is the DNA digestion stage of DNA profiling

Third Stage
Restriction endonucleases cuts up the DNA into smaller fragments at recognition sites (specific sequence of bases).
They must not cut through VNTRS

What is the gel electrophoresis stage of DNA profiling

Final stage (where banding pattern is created)
Splits fragments depending on size because smaller fragments move faster through the gel.
These can be visualised using radioactive or fluorescent bands or probes.

How do you interpret DNA profiles

Patterns can be compared to other profiles
If they are the same, the DNA is likely to have come from the same person.
If they share around 50% of the bases, they are likely to share a close genetic relationship.

Describe the use of DNA profiling in criminal investigations

Can be used to prove guilt/innocence Generated using DNA found in blood, hair or skin cells found at the scene. Mouth swabs may be taken if any suspects Banding profiles are then compared to see if the suspects were at the scene of the crime. This doesn’t always prove guilt though due to possible contamination or because they may have visited the scene days prior to the crime.

Describe the use of DNA profiling in genetic relationships

Can be used in paternity disputes by comparing potential fathers and mothers band profiles to the child’s. Can also be used in immigration cases to prove or disprove family relationships.

Describe the use of DNA profiling in the analysis of disease risk

Can identify individuals at risk of developing certain diseases by looking at VNTRS Certain VNTRS are correlated with an increased risk of disease (tandem repeats of the nucleotides CAG are associated with the risk of Huntingtons disease, risk is certain if over 40 repeats)

What is DNA sequencing?

The process used to determine the precise sequence of nucleotides in a length of DNA (e.g. a gene)

What are the principles of Sanger Sequencing

Involves interrupted PCR followed by gel electrophoresis. Interrupted PCR as Sanger sequencing uses chain terminating nucleotides which are labelled with a radioactive isotope (usually on the phosphate) and is a p32.

What are chain terminating nucleotides?

Modified nucleotides that do not allow the addition of any other nucleotides once they've been incorporated into the chain (they stop DNA synthesis)

What is the process of Sanger Sequencing?

DNA to be sequenced is put in 4 tubes and mixed with primers, DNA polymerase and activated free nucleotides Chain terminating nucleotides for one specific base each are also added to each tube. Tubes are placed into the thermocycler and PCR begins During PCR, chain terminating nucleotides are randomly incorporated into the DNA chains which will stop the DNA synthesis This process is repeated which will result in thousands of DNA fragments which all differ in length by one base

How to read the DNA sequences produced by Sanger sequencing

Fragments are pipetted into separate wells in an agarose gel Gel electrophoresis separates the fragments of DNA according to their length Bands of DNA are visualised as the radioactive chain terminating nucleotides expose photographic paper, this is called an autoradiogram Bands are read off from the bottom to the top

Process of fluorescent Sanger sequence

Carried out in a sequencing machine Instead of radioactive markers, fluorescent markers are used to differentiate each of the four chain terminating nucleotides (only one PCR reaction is needed) The reaction mixture includes DNA fragments, DNA polymerase, primers,activated free nucleotides and low proportion of chain terminating nucleotides Fragments are separated according to length by capillary gel electrophoresis (laser reads fragments as they move fast and detect the colour) Data is recorded as a series of peaks of the different colours and this is known as a chromatogram.