PCR And It’s Role In Diagnostics Flashcards
What is PCR?
An enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process
How many molecules do you have after ‘n’ cycles of a chain reaction?
2^n
At what point is PCR done, and what does it mean?
Melting point of the primer, so under high stringency conditions
What is a synonym for annealing?
Hybridisation of primers
How does PCR amplify specific DNA segments?
The primers hybridise to the chain, are recognised by DNA polymerase and forming the initiation complex around them
What does exponential amplification require?
Two primers that are both complementary
What is the polymerase used in PCR?
DNA dependant DNA polymerase
What is the newly formed strand in PCR called?
Nascent strand
What does DNA dependant DNA polymerase do?
Recognises a specific structure that has a partially double stranded DNA forming an initiation complex with it
How is a partially double stranded DNA structure formed?
By annealing a short single stranded DNA molecule
What is the efficiency of annealing and renaturation dependant upon?
The kinetics of the reactants
What is the DNA dependant DNA polymerase used to do?
To synthesise a new nucleic acid strand by copying a DNA molecule
What can’t DNA dependant DNA polymerase do?
Copy nor make RNA
What does PCR need?
A template strand with an annealed primer
Deoxynucleotide triphosphates
Magnesium ions
A roughly neutral pH
What are the four deoxynucleotide triphosphates?
dATP, dGTP, dCTP, dTTP
What are the three states PCR is dependant on?
Denaturing, annealing and the native state
What is the native state in PCR?
Optimal extension temp and pH for enzyme activity
What must the polymerase be in PCR?
Thermostable
What are the stages (and temperatures) of PCR?
Mix the template, primers, enzyme and reactants
Denature (95C)
Anneal at the melting point of primer
Extend from the 3’ end of the primer (72C)
What are the PCR reaction kinetics dependant on?
Depletion of reactants and acidification of the reaction
What do the graphs of PCR reactions look like?
Sigmoidal linear curve or a straight line log graph with a plateau at the top
What are the diagnostic applications of PCR?
Detecting TB in sputum
Differentiation between closely related organisms like swine flu and human influenza
HIV viral load
COVID testing
How do you measure output of a PCR?
Quantitative or real-time PCR
How does qPCR work?
Using a fluorescent detection of product at the end of each cycle
What is a single nucleotide polymorphism (SNP)?
Single base alteration
What is an example of a single nucleotide polymorphism (SNP)?
Sickle cell anaemia
What are the common applications of single nucleotide polymorphism (SNP) detection?
Antibiotic resistance testing and ID of genetic markers
What are the two approaches to single nucleotide polymorphism (SNP) detection?
High resolution melting and probe based qPCR (allelic discrimination)
How does high resolution melting work?
The melting point of amplified products are used to determine which sequence variant is present
How does allelic discrimination work?
Specific binding of the probe to the amplified region containing the SNP is detected
How is PCR used in forensics and law enforcement?
Parentage or kinship, ID of military of missing people, crime scenes and authentification of biological material
What are short tandem repeats (STRs)?
2-5 bps that are repeated many times at many places in the genome
What does polymorphic mean?
Number of repeats vary between individuals but are similar between siblings and parents
How many short tandem repeats (STRs) does the UK DNA database currently contain?
10
What is the matching probability of the UK DNA database?
1 in 1 billion
When else (not forensic or diagnostics) is PCR used?
For amplifying materials before doing next gen sequencing, manipulating and modifying DNA and for use with recombinant DNA tech
