6.3.4 the polymerase chain reaction

Question 1

define the polymerase chain reaction

Answer 1

biomedical technology in molecular biology which can amplify short length of DNA to thousands of millions of copies

Question 2

who developed the polymerase chain reaction (PCR)

Answer 2

kary mullis (1983) = enables DNA to be analysed

Question 3

what did PCR soon become used for

Answer 3

incorporated into forensic DNA analysis & protocols for analysis of DNA for genetic disease

Question 4

which facts does PCR rely on

Answer 4

• DNA made of 2 antiparakek backbone strands
• each strand of DNA has 5’ end & 3’ end
• DNA grows from 3’ end
• base pairs pair up according to complementary base paring rules

Question 5

how does PCR differ from DNA replication

Answer 5

• only short sequences (up to 10,000 base pairs) can be replicated, not entire chromosomes
• requires addition of primer molecules to begin process
• cycle of heating/cooling required to separate DNA strands, bind primers to trends & for DNA strands to replicate

Question 6

what reaction is PCR

Answer 6

cyclic reaction

Question 7

PCR procedure

Answer 7

1. sample of DNA mixed with DNA nucleotides, primers, magnesium ions & enzyme (taq DNA polymerase)
2. mixture heated to 94-96 degrees breaking hydrogen bonds between complementary nucleotide base pairs = denatures double-stranded DNA into 2 single strands
3. mixture cooled to 68 degrees (so primers can anneal to 1 end of eachh single DNA strand) = gives small section of double-stranded DNA at end of both single-stranded molecules \4. taq DNA polymerase enzyme molecules bind to end (= double stranded)
4. temperature raised to 72 degrees (optimum temp) = keeps DNA as single strands
5. taq DNA polymerase catalyses addition of DNA nucleotides to single-stranded DNA molecules (beginning at end with primer in 5’ to 3’ direction)
6. taq DNA polymerase molecule reaches other end = new double strand of DNA generated
7. process begins again & repeated for many cycles

Question 8

how does the amount of DNA increase (after each round of PCR)

Answer 8

exponentially (1-2-4-8-16-32-64..)

Question 9

applications of PCR

Answer 9

◦ tissue typing = donor/recipient tissues can be typed prior to transplantation to reduce rejection risk
◦ detection of oncogenes = if type of mutation involved in specific patients cancer is found, medication can be better tailored to patient
◦ detecting mutations = sample of DNA analysed for mutation that leads to genetic disease
‣ parents can be tested to see if they carry
recessive allele for certain genes
‣ fetal cells can be obtained from mother’s
bloodstream for prenatal genetic
screening
◦ identifying viral infections = sensitive PCR tests can detect small quantities of viral genome amongst host cells’ DNA (eg. used to verify HIV/hepatitius C infections)
◦ monitoring spread of infectious disease = spread of pathogens though population of wild/domestic animals (or animals to humans) can be monitored & emergence of more virulent subtypes detected
◦ forensic science = small quantities of DNA amplified for DNA profiling, identify criminals or ascertain parentage
◦ research = amplifying DNA from extinct ancient sources (eg. woolly mammoth bones) for analysis/sequencing