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CMB2000 - Essential Biomedical Research > Strand A > Flashcards

Strand A Flashcards

1
Q

What is PCR?

A

It is the amplification of DNA
Based on DNA polymerase being able to synthesise a new strand
Starts with single stranded DNA
Taq polymerase used
Exponential increase in strands

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2
Q

What ingredients are used in eukaryotic replication?

A

Template DNA
40 or more proteins
Helicase
Primase
Polymerases
Nuclease
Ligase
ssDNA binding proteins (protects DNA)
Sliding clamps (polymerase doesnt fall off)

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3
Q

Why is PCR used?

A

Sensitive - amplify as little as one molecule of DNA
Specific - amplify a unique target sequence (temperature and Mg2+ specific)
Cheap
Rapid - few hours
Robust - DNA is very stable can be amplified from old degraded samples

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4
Q

What is typically in a PCR reaction tube?

A

Template dsDNA
2 primers to prime synthesis
Polymerase to copy the template
dNTPs
Magnesium as a co-factor
Buffer to maintain pH and provide necessary salt

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5
Q

How do nucleotides act as building blocks?

A

They contain functional groups that form bonds to create the copies

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6
Q

What is the function of taq polymerase?

A

Copy DNA accurately
Used for: synthesis, proof reading and primer removal

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7
Q

How are primers designed?

A

2 primers (forward and reverse)
Single stranded DNA
Length 18-24 (too long = hybridise too slowly, too short = not specific)
40-60% G/C content
Start and end with 1-2 G/C pairs
Melting temperate 50-60C
3’ end must be complementary to template DNA
Primer pairs should not have complementary regions

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8
Q

What is the function of magnesium in PCR?

A

It acts as a co-factor and a non-protein component that enables the activity of the catalysis
It enhances the enzymatic activity supporting DNA application

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9
Q

What is the function of a buffer in PCR?

A

Optimal pH is 8-9.5
Tris HCl
Potassium ions - promotes annealing
May be replaced by ammonium sulphate (destabilises base pairing bonds)

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10
Q

How many cycles are in PCR?

A

~30

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11
Q

What PCR products can be identified on a gel?

A

Molecular weight markers
PCR products
Primers
Template

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12
Q

What is an example of PCR in biotechnology?

A

Manipulate DNA - artificial fragments and then introduce into host cells and gets incorporated into genome
Knock out genes
Fuse host proteins with green fluorescent

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13
Q

How is reverse transcriptase PCR carried out?

A

Converts RNA to cDNA, use reverse transcriptase that converts RNA to DNA
Amplify DNA by PCR

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14
Q

What can be the source of the RNA for RT PCR?

A

Gene expression (mRNA)
RNA virus

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15
Q

What are the differences between end point PCR and real time PCR (qPCR)?

A

End point: Cheap
Semi-quantitive (band intensity)
Sequencing, genotyping and cloning
See results at the end
Real time: More expensive
Quantity PCR proportional to amount of template
Quantification gene expression, microarray verification, quality control, assay validation etc
Measures at exponential phase (more precise)

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16
Q

What are the different reagents used in qPCR?

A

SYBR green and TaqMan

17
Q

How does SYBR green work?

A

Binds to the grooves in the DNA which increases the fluorescence

18
Q

How does TaqMan work?

A

Probes are used with a fluorescence reporter and quencher

19
Q

How does fluorescence quantify qPCR?

A

Using at threshold line at the cycle threshold (Ct)
A lower Ct = more cDNA in sample
Housekeeping gene
Standard curve
Standard points

20
Q

What are housekeeping genes?

A

They are genes with a constant level of expression which are essential to support validity of qPCR
Confirms RNA extraction was good and efficient
Supports conclusions of expression levels

21
Q

What is Nancy520 and how does it work?

A

Intercalating agent that goes between base pairs to allow the detection of PCR products

22
Q

Why is PCR clinically valuable?

A

Sensitive
Specific (unique targets)
Relatively cheap
Rapid (only a few hours)
Robust - DNA is very stable

23
Q

What are the different uses of PCR when determining diagnosis and prognosis?

A

Genotyping the patient - genetic traits, carriers, tissue matching and predicting response to drugs
Genotyping the pathogen - diagnosis of species and strain
Phenotyping the disease - measuring disease progression and disease severity

24
Q

What is genotyping the patient?

A

Detecting which alleles an individual carries for a specific gene

25
Q

What can sources of DNA be to genotype a patient?

A

Blood
Hair
Buccal smear
Cells from amniotic fluid

26
Q

What are the 2 PCR based techniques for genotyping an individual?

A

PCR-RFLP (restriction fragment polymorphism)
ARMS-PCR (amplification refractory mutation system)

27
Q

How does PCR-RFLP work?

A

Identifies allelic variants based on presence/absence of restriction site

28
Q

What is the process of PCR-RFLP?

A

Allele 1 - healthy, Allele 2 - diseased
1 - no restriction site, 2 - has restriction site
Results during electrophoresis produce a heterozygous genotype

29
Q

What are the advantages and disadvantages of PCR-RFLP?

A

Advantages: Cheap
Easy design
Applied to microindels and SNPs
Simple resources
Commonly used technique
Disadvantages:
Only possible with known RE site
RE expensive
Single nucleotide variation
Hands on and time consuming
Not suitable for high throughput

30
Q

How does ARMS-PCR work?

A

Detects allelic variants using allele specific primers (only point mutations)

31
Q

What is the process of ARMS-PCR?

A

2 Allele specific primers and conserved provers are used to amplify the DNA
Amplification takes place on which provides no amplification with one of the alleles but not the other
1 band will show up one will not

32
Q

What are the differences between RFLP and ARMS?

A

RFLP: Uses locus specific primers
Relies on presence or absence of a restriction site to distinguish between variants
ARMS: Allele specific primers
Relies on stringency of PCR to distinguish
Alternative Tetra Primer ARMS-PCR, additional non-allele specific primers

33
Q

What is genotyping the pathogen?

A

Identifying the species and strain of an infectious pathogen

34
Q

Where can DNA/RNA be obtained to genotype a pathogen?

A

Blood
Sputum
Urine
Faeces
Skin swab
Tissue biopsy

35
Q

What is the advantage of using PCR to diagnose microbes?

A

Vs microscopy: sensitive - detect single copies
Specific - identifies species and strain
Vs culture: sensitive - no need culture
PCR is quick
Vs patient antibody response: detects DNA/RNA therefore not dependent on immune response

36
Q

How is a disease phenotypes?

A

To allow a snapshot of the disease status, severity or progression

37
Q

What is a key technique to phenotype a disease?

A

Quantitive PCR - measuring the abundance of DNA/RNA in a clinical sample

38
Q

What does ARMS-PCR stand for?

A

Amplification Refractory Mutation System

CMB2000 - Essential Biomedical Research (2 decks)

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