Biology - Review - 13

Question 1

Why is DNA ‘amplified’?

Answer 1

DNA is often found or extracted in trace amounts and requires amplification to produce an adequate sample for scientists to work with.

Question 2

What reaction is used to Amplify DNA?

Answer 2

DNA amplification uses the polymerase chain reaction (PCR) to rapidly increase the amount of DNA

(creating identical copies of the DNA sample).

Question 3

What are Polymerases?

Answer 3

Polymerases are enzymes that create polymers of nucleic acids (DNA and RNA). DNA polymerase acts to assemble DNA molecules, and RNA polymerase acts to assemble RNA molecules.

Question 4

What is Taq polymerase?

Answer 4

Taq polymerase is a heat-resistant DNA polymerase used in PCR.

Question 5

What is PCR?

Answer 5

PCR is a technique that amplifies DNA exponentially to create millions of identical copies of the DNA from a very small sample.

Question 6

How is the PCR technique done?

Answer 6

The sample plus two primers, free nucleotides and Taq polymerase are added to a test tube and the test tube is placed in a thermocycler. The thermocyder alters the temperature in pre-programmed stages to enable a three-step process to be carried out:

• Denaturation
• Annealing
• Extension
  The three steps of the PCR cycle are repeated many times (up to 50 cycles) to create large quantities of the DNA sample.

Question 7

What is the ‘Denaturing’ step of the PCR technique?

Answer 7

PCR Technique

Denaturation—separation of the two strands of DNA at 95*C

Question 8

What is the ‘Annealing’ step of the PCR technique?

Answer 8

PCR Technique

annealing—joining at 50-60 *C of a primer to each strand to create a starting point for Taq polymerase to copy

Question 9

What is the ‘Extension’ step of the PCR technique?

Answer 9

PCR Technique

Extension—Taq polymerase adds free nucleotides to the DNA to create new. identical DNA at 72 °C.

Question 10

What is Gel electophoresis?

What is it, and why is it used?

Answer 10

Gel electrophoresis is a technique that separates fragments of DNA by length, using their negative charge.

It can be used to:

• Identify species e.g. of virus contamination in water
• Identify mutations e.g. cystic fibrosis (recessive mutation) so siblings of can be tested to see if they are carriers.

Question 11

What is the technique for Gel Electrophoresis?

6 steps

Answer 11

1. Each DNA sample is loaded into a different well in an agarose gel.
2. The wells are located at the negative end of a gel electrophoresis chamber.
3. A DNA ladder is also added to the reference well.
4. The agarose gel is submerged in a buffer solution and an electric field is generated.
5. DNA migrates through pores in the gel towards the positive terminal due to it’s negative charge.
6. Smaller fragments move faster and therefore further through the gel than longer fragments in a given time.

Question 12

How is Gel Electrophoresis assessed?

Answer 12

• After the electic field is switched off the gel is viewed to identify the number and size of DNA fragments.
• DNA is visible as bands in a stained gel.
• The location of a band directly correlates with its specific length so visible fragments can be identified and measured by comparing their position to the ‘DNA ladder’.

Question 13

What are Restriction Enzymes?

Answer 13

Restriction enzymes are enzymes that cut DNA at particular recognition sites.

• Sticky-end restriction enzymes leave fragments with overhanging ends that have exposed bases.
• Blunt-end restriction enzymes cut DNA to leave flat-ended fragments.

Question 14

What are Ligase enzymes?

Answer 14

Ligase enzymes permanently join fragments of DNA or RNA together in a process called ligation. DNA ligase joins DNA fragments. RNA ligase joins RNA fragments.

Question 15

What are Plasmids?

Answer 15

Plasmids are small, circular pieces of double- stranded DNA found in bacterial cells. They replicate independently of the bacteria’s chromosomal DNA.

Question 16

What are Recombinant Plasmids?

Answer 16

Recombinant plasmids are plasmids that have had target DNA inserted into them. The same sticky-end or blunt-end restriction enzyme is used to cut both the targeted gene and the plasmid, and then DNA ligase is used to permanently join the two together.

Question 17

What are plasmids with antibiotic resistance used for?

Answer 17

Plasmids with antibiotic resistance are generally used to enable identification of bacterial transformation later on, as only bacterial cells containing these plasmids will survive when grown in cultures containing the antibiotic

Question 18

What is a reporter gene?

Answer 18

A gene that produces an identifiable phenotype, such as a coloured product or fluorescence, may be used as a reporter gene to identify transformed cells.

Question 19

How is a reporter gene used?

Answer 19

Disruption of a reporter gene by insertion of the target gene can indicate successful bacterial transformation, as the gene no longer functions.

For example, if the lacZ gene is disrupted by insertion of a target gene, the bacterial cell cannot break down the X-gal indicator present in the agar medium. Colonies appear white instead of blue.

Positive expression of a reporter gene, such as green fluorescent protein, is also used to indicate successful bacterial transformation.

Question 20

What are the steps in bacterial transformation?

Answer 20

There are three steps in bacterial transformation:

• Gene uptake
• Selection of transformed bacteria
• Identification of transformed colonies

Question 21

What is ‘Gene uptake’ in bacterial transformation?

Answer 21

Gene uptake

Bacterial cells are induced to take up the recombinant plasmids either by heat shock or electroporation methods. Very few bacterial cells will be transformed (and some of these will contain non-recombinant plasmids).

Question 22

What is ‘selection’ in bacterial transformation?

Answer 22

Selection of transformed bacteria

The bacteria are grown in the presence of an antibiotic. Bacterial cells that have been transformed will survive, as the gene for antibiotic resistance is located on the plasmid.

Question 23

What is ‘Identification of transformed colonies’ in bacterial transformation?

Answer 23

Identification of transformed colonies

Transformed bacteria containing recombinant plasmids are identified by the reporter gene in the recombinant plasmid. For example, if the lacZ gene is the identifying gene, bacterial colonies will be white rather than blue as the /acZ gene will be disrupted by the insertion of the target DNA. and will no longer be functional.