PCR And Diagnostics Flashcards
What is the definition of PCR?
→ An enzyme based method to specifically amplify segments of DNA using thermal DNA polymerase in a cyclical process
What type of increase is there in PCR?
→ Exponential
What does the specificity stem from in PCR?
→The complementarity of the primers
→high stringency conditions under which only perfectly matched duplex will form
Under what conditions is PCR specific?
→ If annealing is undertaken at the melting point of the primers
What does high stringency mean?
→ High temperature
If you want to amplify a segment bound by a known sequence how do you do this?
→ Primers that are complementary to these ends
What does DNA polymerase recognize?
→ A specific structure consisting of a partially double stranded DNA forming an initiation complex
How is a partially double stranded structure formed?
→ By annealing a short single stranded DNA molecule to a denatured single stranded molecule
How do you prevent annealing and renaturation happening at the same time?
→ Driven by having an excess of the primer
What kind of polymerase is used in PCR?
→ DNA dependent DNA polymerase
How does DNA dependent DNA polymerase work?
→ Synthesizes a new nucleic acid by copying a DNA molecule
How can RNA be amplified?
→ It must be copied into DNA or cDNA
→ By reverse transcriptase
What are the 4 requirements of DNA polymerase to work?
→ Template strand with a primer
→ Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
→ Mg2+ ions
→ Roughly neutral pH
What are the three states that PCR depends on?
→ Denaturation
→ Annealing
→ Native state at the optimal extension temperature and pH for enzyme activity
What does PCR need to work?
→ Multiple rounds of extreme heating and cooling
What property must the DNA polymerase have for PCR to work?
→ Thermostable
What does thermostability mean?
→ Ability to retain activity upon repeated heating to temperatures that would destroy most enzymes
What species of polymerase is used in PCR?
→ Thermophilus Aquaticus
What are the 6 steps of PCR?
1) All reactants : template, primers and enzymes are mixed
2) Denaturation at 95 degrees
3) The mixture is cooled to the primer Tm and they bind to the template strand
4) The temperature is then changed to 72 degrees, a temperature which is optimal for the enzyme to work
5) An initiation complex is formed which elongates from the 3’ end which creates a second strand
6) This is repeated multiple times
What are the kinetics of the reaction dependent on?
→ Depletion of reactants
→ Acidification of reaction
Why is there acidification?
→ H+ ions get produced as dNTPs are added
→ Pyrophosphate is produced
What are 3 ways PCR used in diagnostics?
→ Presence or absence calling TB - detection in sputum
→ Differentiating between closely related organisms ( swine flu vs human influenza)
→ Treatment starting (HIV viral load)
What is the main limitation of PCR?
→ The end point is the same irrespective of the starting concentration
→ Limited by the amount of DNA polymerase
How does real time PCR work?
→ Fluorescent detection of the amplification
What is real time PCR for?
→ Quantifying the amount of a target DNA molecule in the sample
What are SNPs?
→variations in the DNA sequence at particular locations, called single nucleotide polymorphisms
→ Difference of a single nucleotide
What does SNP detection depend on?
→ Differences in melting temperature (Tm)
→ conferred due to the nucleotide composition
What is SNP detection used for?
→ Antibiotic testing - TB and other organisms
→ Identification of genetic markers - drug sensitivity
What is high resolution melting?
→ Tm of the amplified product is used to determine which sequence is present
What is the probe based version of qPCR?
→ Specific binding of the probe to the amplified region containing the SNP is detected
What are the 4 uses of PCR in forensics?
→ Parentage or kinship - immigration and inheritance
→ Identification - military casualties, missing persons or environmental disasters
→ Matching two sources - crime scenes
→ Authentication of biological material - cell lines, purity of food
What are STRs?
→ STRs are 2-5 or more bases repeated at specific locations in the genome
What do STRs provide?
→ A pattern of uniquely sized products
→ Molecular barcode
What does the UK DNA database consist of?
→ 10 STRs
What does forensic identification use?
→ STRs
How is PCR designed so it will span many STRs?
→ Multiple sets of labelled primers
What is another application of PCR in research?
→ Manipulating and modifying DNA
→ Introducing mutations into a sequence
What are primers?
complementary to sequences at the ends of the amplicon and are able form a duplex by hybridising to them
What does the exponential amplification depend on?
having two primers each complementary to one of the two strands
How do we make PCR quantitative?
place a threshold on the measurement of product and compare this to a standard curve of known concentration
What are the two approaches to SNP detection?
High resolution melting (HRM)
Probe based version of qPCR
What are other uses of PCR?
→Amplifying material prior to:
Next generation sequencing eg.
Isolating individual segments of DNA prior to cloning or sequencing
→Manipulating and modifying DNA
→Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors
→developing recombinant vaccines
