6.3.4: The polymerase chain reaction

Question 1

What is polymerase chain reaction (PCR)?

Answer 1

A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies.

Question 2

What does PCR rely on?

Answer 2

The facts that:

• DNA is made of two antiparallel backbone strands.
• Each strand of DNA has a 5’ end and a 3’ end.
• DNA grows only from the 3’ end.
• Base pairs pair up according to complimentary base pairing rules (AT, CG)

Question 3

How does PCR differ from DNA replication?

Answer 3

• Only short sequences, of up to 10 000 base pairs, of DNA can be replicated, not entire chromosomes.
• It requires the addition of primer molecules to make the process start.
• The cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated.

Question 4

What type of DNA polymerase do we use in PCR and why?

Answer 4

• We use DNA polymerase from the thermophilic bacterium Thermophilus aquaticus.
• This enzyme is called Taq polymerase and is heat stable at higher temperatures.

Question 5

What are the steps involved in PCR?

Step 1:

Answer 5

The sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase.

Question 6

What are the steps involved in PCR?
Step 1: The sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase.

Step 2:

Answer 6

The mixture is heated to around 94-96 degrees celsius to break the hydrogen bonds between complementary nucleotide base pairs and thus denature the double strand DNA into two single strands of DNA.

Question 7

What are the steps involved in PCR?
Step 2: The mixture is heated to around 94-96 degrees celsius to break the hydrogen bonds between complementary nucleotide base pairs and thus denature the double strand DNA into two single strands of DNA.

Step 3:

Answer 7

The mixture is cooled to around 68 degrees celsius so that the primers can anneal (bind by hydrogen bonding) to one end of each single strand of DNA. This gives a small section of double-stranded DNA at the end of each single-stranded molecule.

Question 8

What are the steps involved in PCR?
Step 3: The mixture is cooled to around 68 degrees celsius so that the primers can anneal (bind by hydrogen bonding) to one end of each single strand of DNA. This gives a small section of double-stranded DNA at the end of each single-stranded molecule.

Step 4:

Answer 8

The Taq DNA polymerase enzyme molecule can now bind to the end where there is a double-stranded DNA. Taq polymerase is obtained from a bacterium that lives at high temperatures; 72 degrees celsius is the optimum temperature for this enzyme.

Question 9

What are the steps involved in PCR?
Step 4: The Taq DNA polymerase enzyme molecule can now bind to the end where there is a double-stranded DNA. Taq polymerase is obtained from a bacterium that lives at high temperatures; 72 degrees celsius is the optimum temperature for this enzyme.

Step 5:

Answer 9

The temperature is raised to 72 degrees celsius, which keeps the DNA s single strands.

Question 10

What are the steps involved in PCR?
Step 5: The temperature is raised to 72 degrees celsius, which keeps the DNA s single strands.

Step 6:

Answer 10

The Taq DNA polymerase catalysed the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with a primer and proceeding int the 5’ 3’ direction.

Question 11

What are the steps involved in PCR?
Step 6: The Taq DNA polymerase catalysed the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with a primer and proceeding int the 5’ 3’ direction.

Step 7:

Answer 11

When the Taq DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated.

Question 12

What are the steps involved in PCR?
Step 7: When the Taq DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated.

Step 8:

Answer 12

Then the process begins again and is repeated for many cycles

Question 13

How does the amount of DNA increase in PCR?

Answer 13

It increases exponentially (doubles each cycle)

Question 14

How is PCR used in tissue typing?

Answer 14

Donor and recipient tissues can be typed prior to transplantation to reduce the risk of rejection of the transplant.

Question 15

How is PCR used in the detection of oncogenes?

Answer 15

If the type of mutation involved in a specific patient’s cancer is found, then the medication may be better tailored to that patient.

Question 16

How is PCR used in detecting mutations?

Answer 16

• A sample of DNA is analysed for the presence of a mutation that leads to a genetic disease.
• Parents can be tested to see if they carry a recessive allele for a particular gene.

Question 17

How is prenatal genetic testing carried out (in both natural and IVF births)?

Answer 17

• Fetal cells may be obtained from the mother’s bloodstream for prenatal genetic screening
• During IVF treatment, one cell from an eight-cell embryo can be used to analyse the DNA before implantation.

Question 18

How is PCR used in identifying viral infections?

Answer 18

Sensitive PCR tests can detect small quantities if viral genome amongst the host cells’ DNA. This can be used to verify, for example, HIV or hepatitis C infections.

Question 19

How is PCR used to monitor the spread of infectious disease?

Answer 19

-The spread of pathogens through a population of wild or domestic animals, or from animals to human populations, can be monitored, and the emergence of new more virulent sub-types can be detected.

Question 20

How is PCR used in forensic science?

Answer 20

-Small quantities of DNA can be amplified for DNA profiling to identify criminals or to ascertain parentage.

Question 21

How is PCR used in research?

Answer 21

• Amplifying DNA from extinct ancient sources such as Neanderthal or woolly mammoth bones, for analysis and sequencing.
• In extinct organisms, tissues or cells can be analysed to find out which genes are switched on or off.