DEPTH STUDY

Question 1

Polymerase Chain Reaction (PCR)

Lesson 1

Answer 1

A biotechnology technique used to make copies of a piece of DNA (known as DNA amplification) without a cell.

It is performed on a sample of DNA mixed with primers and other solutions in a tube that is placed in a PCR machine.

The following steps are performed:

1. Denaturation
2. Annealing
3. DNA Synthesis

Question 2

1) Denaturation

Lesson 1 - PCR

Answer 2

The solution is heated to 94-96°C to break the hydrogen bonds between complementary DNA strands that thus separate the two.

Question 3

2) Annealing

Lesson 1 - PCR

Answer 3

The solution is cooled to approximately 68°C, allowing the primers to bind to the target DNA sequence and initiate DNA polymerisation (the construction of complementary DNA strands to the template strands)

Question 4

3) DNA Synthesis

Lesson 1 - PCR

Answer 4

Taq polymerase enzymes in the solution move along the two original template strands and join the free nucleotides together to form new DNA strands.

Tag polymerase is heat resistant as any other enzyme would denature.

Question 5

Primers

Lesson 1

Answer 5

The Facts:

• DNA polymerase can only add free nucleotides to the 3’ end of an existing strand
• DNA primase adds a complemetary RNA primer to the reading template of both the leading and lagging strands where DNA polymerase can extend it.
• RNA primers are reoved and replaced with new DNA that fills the gaps

Question 6

DNA Sequencing

Lesson 1

Answer 6

The process of determining the exact nucleotide sequence in DNA through techniques such as the Sanger, Massively Parallel Sequencing and newer Nanopore Sequencing.

The process is as follows:

1. DNA is mixed in a solution of different types of terminating nucleotides that are tagged with different coloured fluorescent dyes.
2. After PCR is completed, the DNA mixture is placed into an apparatus that performs capillary electrophoresis to separate the lengths of fragments.
3. A laser beam is shone at the fragments to identify the terminating bases, which are analysed by a computer program to reveal the DNA sequence.

Question 7

DNA Profiling

Lesson 2

Answer 7

The process of determining an individual’s DNA characteristics.

Although the DNA sequence among humans is mostly similar, non-coding sections of DNA can vary widely, so these genetic markers are used for individual identification.

Question 8

1) Extracting DNA from the Sample

Answer 8

1. Collect cells
   
   • This is most commonly done by swabbing cheek cells from inside an individual’s mouth.
   • It is then placed in a tube with some lysis solution.
2. Burst cells to release DNA
   
   • The tube is placed in a warm water bath. The detergent in the lysis solution disrupts the cell membrane and causes it to burst, while the enzyme proteinase K cuts apart the histones to release the DNA.
3. Separate DNA from proteins and debris
   
   • A concentrated salt solution is added to the tube, which causes the cellular debris to clump together.
   • The tube is placed into a mini centrifuge to separate the debris from the DNA solution. The centrifuge must be balanced to work so a tube of water may be inserted opposite.
   • A micropipette is used to separate the top liquid containing the DNA strands and put it into another tube.
4. Isolate concentrated DNA
   
   • Isopropyl alcohol is added to the tube, and it is inverted several times. DNA is insoluble in isopropyl alcohol, so it clumps.
   • The tube is placed into a mini centrifuge to separate DNA from the liquid, which can then be pipetted out of the tube.

Question 9

2) PCR

Lesson 2 - DNA Profiling

Answer 9

Adding the DNA of interest to the PCR allows each STR to amplify, making it easier for study.

Question 10

3) Electrophoresis

Lesson 2 - DNA Profiling

Answer 10

A standard electrophoresis gel or capillary electrophoresis can be used to detect the differences in size of the STRS. A computer registers any peaks in size.

Question 11

Genetic Markers

Lesson 2

Answer 11

Any piece of DNA that can be analysed using sequencing techniques to identify individuals or species.

Common features include:

• Location - the loci is often known within non-coding regions, meaning they can be used as tags
• Polymorphic - there has to be high variation

Examples include:

• Single nucleotide polymorphisms (SNPs)
• Short tandem repeats (STRs)

Question 12

Short Tandem Repeats (STRs)

Lesson 2 - Genetic Markers

Answer 12

Short sections of DNA with repeating nucleotides, of which there are thousands in the human genome.

• Each person has two copies of every STR as they inherit one from each parent. If both are the same, they are homozygous and vice versa.
• The size of STRs at different sites can be compared to another sample or a database.
• Sine there are thousands of STR sites and they are highly variable, it is extremely unlikely for two individuals to match across multiple sites.

Question 13

Ethical Considerations of DNA Profiling

Lesson 2

Answer 13

• Who owns the information?
• Who can access the information?

Question 14

Electrophoresis

Lesson 2

Answer 14

To separate the DNA fragments based on size, the following procedure is performed:

1. Agarose gel is immersed in a buffered salt solution in an electrophoresis box to stabilise pH, keep it moist, and make it electrically conductive.
2. The DNA is mixed with a loading buffer that contains fluorescent dye to attach to the fragments.
3. DNA samples are placed into prepared wells in the gel.
4. An electrical current is applied to the gel so that the fragments will migrate through the gel to the positive terminal as the DNA’s phosphate group is negative.
5. Longer fragments are heavier and will mover slower than the shorter, so fragments become increasingly separated through the gel.
6. After a period of time, the gel is removed from the salt solution and placed in a bath of DNA staining solution of ethidium bromide, which fits between the rungs of the DNA ladder.
7. Place the gel into a UV light box to observe the fragments, which can then be estimated by comparing them to the standard DNA ladder.

Question 15

Restriction Enzymes/Endonucleases

Lesson 2 - Electrophoresis

Answer 15

Naturally occurring proteins found in many prokaryotic cells such as bacterial cells.

• Their biological purpose is to protect the cell from foreign and pathogenic DNA and RNA molecules, which it can cut up into nonfunctional fragments.
• In the laboratory, biochemists can extract and copy these restriction enzymes and use them for a variety of purposes

When a restriction enzyme cuts…

• It cleaves the phosphodiester bonds at palindromic sequences, creating twofold, rotational symmetry in the fragments.
• This produces sticky ends that are useful for recombinant DNA or some are blunt cutters, which can be rejoined with DNA ligase but this is more difficult to position.

Question 16

Lesson 3

Question 17

DNA Cloning

Lesson 4

Answer 17

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. It is performed through the following steps:

1. Ligation
2. Bacteria Transformation

Question 18

1) Ligation

Lesson 4 - DNA Cloning

Answer 18

1. Separately cut the gene fragment and the plasmid with the same restriction enzyme to create complementary sticky ends.
2. Take the gene fragment and the linearised (opened up) plasmid and combine them along with DNA ligase according to their complementary base pairing.
3. This produces a molecule of recombinant DNA or a recombinant plasmid, which is DNA assembled out of fragments from multiple sources.

Question 19

Bacteria Transformation