30. PCR

Question 1

what is PCR?

Answer 1

• polymerase chain reaction
  – based off the ability of DNA polymerase to synthesise a complementary strand of DNA
• PCR uses oligonucleotide primers
  – short sequences complementary to outer regions of a known sequence to be amplified
• complementary paired primers
  – starting point for DNA polymerase to add base pairs complementary to ‘primed’ template strands

Question 2

what does PCR require?

Answer 2

• template DNA
  – DNA containing regions of DNA to be amplified
• primers
  – synthetic strands of DNA (18-25 bp length)
• DNA polymerase
  – enzyme sequentially adding nucleotides complementary to target DNA on 3’ end of primer sequence
• nucleotides (dNTPs)
  – dATP, dTTP, dGTP, dCTP
  – building blocks for synthesis of new DNA strands
• MgCl / MgSO4
  – Mg2+ co-factor for DNA polymerase to function efficiently
  – affects specificity of primer binding, nautralising negative chage of phosphate groups on DNA backbone. Makes DNA strands repel each other, thus primer stronger in presence of Mg2+ but may lead to primers binding to non-specific templates
• thermocycle (PCR machine) / water bath
  – PCR relies on cycling between approx. 50 and 94 Celcius

Question 3

what DNA polymerase is used in PCR?

Answer 3

• Taq polymerase
  – most commonly used, commercially available
  – isolated from Thermus aquaticus (bacterium from Yellowstone National Park springs)
• Pyrococcus furiosus (Pfu polymerase)
  – higher fidelity (proofreading capacity)
• polymerases
  – have proof reading capabilities 3’-5’ direction
• can remove incorrectly added base and replace with correct
• some DNA polymerases have greater proofreading capability
  – or have been genetically enhances

Question 4

how does PCR primer design work?

Answer 4

• good primers key for successful PCR
• no priming = no 3’ ends for DNA polymerase to add base pairs

Question 5

what do primers need to be?

Answer 5

• specific to DNA sequence amplified
• melting temperature compatible
• GC content
  – 3’ clamp properties (GC content)
  – G=C; A=T (H bonds)
• avoid hairpins / primer-dimers (primer-primer interactions)
  – result in reduce number of primers for reaction

Question 6

what are primer dimers?

Answer 6

• formed when two primers designed for amplification of specific DNA sequence have degree of sequence complementarity
  – allows annealing of two primers to each other
• following syntheis of complementary DNA strands for annealing event
  – sequences become template for subsequent PCR cycles
• results in amplification of primer-dimer rather than desired template

Question 7

what are the steps of PCR?

Answer 7

• denaturation
  – 94-99 Celcius
  – 10-30 sec
• annealing
  – 50+ Celcius
  – depends on annealing T of primers
• extention
  – 72 Celcius
  – 30-180 sec
  – depending on polymerase, processivity of enzyme and length of template amplified (general polymerases 1000 bp/minute

Question 8

what is the purpose of agarose gel electrophoresis?

Answer 8

• agarose gels
  – allow for use of separate DNA fragments by size once electrical current applied
• DNA negative charged
  – thus, will migrate from negative to positive electrodes
• once PCR completed
  – amplified DNA mixed with loading dye
• following electrophoresis
  – DNA visualised by staining ethidium bromide (intercalates bp of double helix, then visualised under UV light)

Question 9

what does loading dye consist of?

Answer 9

• glycerol
  – increase densityof sample
  – allow to drop to bottom of well made in gel during casting
• colour dye
  – allow for tracking movement of sample through gel

Question 10

what is reverse transcription PCR?

Answer 10

• RNA used as template
  – produce cDNA (complementary DNA) using reverse transcriptase
• reverse transcriptase is enzyme retroviruses use to convert their RNA genomes to DNA prior to infection of host cells’ genomes
• PCR used to produce double stranded DNA from template cDNA strand

Question 11

what is real time PCR?

Answer 11

• quantitative PCR
  – target DNA is quantified simultaneously amplifying it
• can detect amplified product as reaction progresses
  – rather than at the end (conventional PCR)
• achieved by performing DNA amplification in presence of DNA bidning dyes
  – bind to double stranded sequence (like SYBR green)

Question 12

what are the applications of PCR?

Answer 12

• presence of specific viruses / bacteria can be tested for in blood sample from which DNA extracted
• viral loads (eg. HIV) can be determine using quantitative PCR
• identification of human remains for forensics
  – identifying criminals when mixed DNA samples present at crime scene