Biotechnology

Question 1

What does PCR do?

Answer 1

• Replicates and amplifies small amounts of DNA.
• This can then be used for detecting genetic diseases, finding out who the parent is, detecting mutations, investigating evolutionary relatedness of species.
• Mimics the process of DNA replication which occurs just before mitosis.

Question 2

What do you need for PCR?

Answer 2

• DNA sample - the one being copied.
• Primer - to indicate and direct the polymerase where to start.
• Free nucleotides - to be able to be added to the new DNA strand.
• Taq polymerase - can withstand heat better than normal polymerase.

Question 3

Describe the steps of PCR.

Answer 3

1. Denaturation - 95°C, the heat breaks hydrogen bonds, separates two strands (like helicase).
2. Annealing - 50-60°C, primers attach to the single strands of DNA complementarily.
3. Extension - 72°C, Taq polymerase binds to primers and begins adding free nucleotides to the open strands, creating a new complementary strand of DNA.

Question 4

How does gel electrophoresis work?

Answer 4

• Separation of DNA strands based on their lengths of nucleotides.

1. DNA is fragmented by restriction enzymes.
2. It is placed into an agarose gel into a well by a micropipette.
3. The power source is activated and the DNA (being negatively charged) begins to move towards the positive electrode.
4. The farther the DNA goes, the shorter the strand is (lattice movement).
5. An image of the bands (DNA profile) is then created which can then be used to compare to other banding patterns.

Question 5

What is DNA sequencing?

Answer 5

• The discovery of the exact order of a DNA sequence.
• Done by the Sanger method.
• Identifies any mutated alleles, genetic diseases, and any similarities and differences between organisms.

Question 6

How does DNA sequencing differ to a DNA profile?

Answer 6

• A DNA profile needs to be compared against others and doesn’t show the exact sequence of DNA whereas DNA sequencing does.

Question 7

What is a dideoxyribonucleotide (ddNTP)?

Answer 7

• A synthetic nucleotide that prevents extensions of a DNA strand.
• Lacks an OH (hydroxyl) group for the next nucleotide to attach onto.
• Randomly attaches to the strand of DNA, stopping the sequence at any point in time.

Question 8

Alright… how does Sanger Sequencing work?

Answer 8

• One needs 4 of the same unknown single-stranded sequences of DNA, four primers, four lots of DNA polymerase, and lots of free nucleotides.
• Each test tube then receives one different ddNTP.

1. The polymerase begins to make new strands of DNA by adding free nucleotides. It continues to do this until a ddNTP is added.
2. The ddNTP stops the process because nothing else can be added onto the missing OH group of it. All the DNA fragments are different lengths.
3. Then, put it in the agarose gel and send it through gel electrophoresis where the banding patterns will be created.
4. This will then be able to be read from the bottom up in line with the other nucleotides, observing the sequence in doing so.

Question 9

What is tissue engineering?

Answer 9

• process of tissue regeneration which eliminates all possibility of tissue rejection.
• healthy stem cells are taken from a patient and embedded and grown on a man-made porous scaffolding.
• the scaffold is then implemented into the patient which then degrades, leaving healthy functional tissue.
• since the tissue is made from the patient’s stem cells, there are no non-self antigens, so the tissue grows in the body.
• tries to eliminate the need for organ transplants by restoring the healthy tissue.

Question 10

What is gene therapy?

Answer 10

• the treatment of genetic disorders by replacing or supplementing defective genes with normal, functioning genes.
• can replace a mutated gene with a healthy copy, fix mutated genes, insert new genes.
• uses a vector to insert the new DNA which then is incorporated by the nucleus to undergo its processes to produce the desired protein.