2. DNA manipulation

Question 1

Describe the denaturing phase of polymerase chain reaction

Answer 1

Denaturing - Heat DNA to 90oC to separate the DNA into single strands by breaking hydrogen bonds between complementary bases.

Question 2

Describe the primer annealing phase of the polymerase chain reaction

Answer 2

DNA Primer Annealing - Cool to approximately 50oC to attach/anneal DNA primers to the single-stranded DNA.

Question 3

Describe the extension phase of the polymerase chain reaction

Answer 3

Extension- Heated to approximately 72oC - DNA Taq polymerase copies the single DNA strands in a 3’ to 5’ direction using complementary base pairing.

Question 4

State how many times the polymerase chain reaction is completed

Answer 4

35+ times

Question 5

State the purpose of the polymerase chain reaction

Answer 5

To amplify the DNA so that there is enough to analyse

Question 6

Name the fours stages of PCR

Answer 6

Denature
Primers Anneal
Extension
Repeat 35+

Question 7

State the three temperatures of PCR

Answer 7

90 C
50 C
72 C

Question 8

Outline the four stages of PCR

Answer 8

Denaturing - Heat DNA to approximately 90C to separate the DNA strands.
Primer annealing - Cool to attach approximately 50C to attach primers at the 3’ end of each DNA strand.
Extension (elongation) - Heat to 70C so Taq polymerase can copy strands in a 3’to 5’ direction.
Repeats - Process is repeated 35 times.

Question 9

Reverse transcriptase is a

Answer 9

enzyme that copies mRNA into copy DNA

Question 10

DNA primers are

Answer 10

Short single-stranded DNA fragments that attach to DNA to allow the binding of DNA taq polymerase

Question 11

State the funciton of an endonuclease

Answer 11

Cut DNA at a specific recognition site

Question 12

Describe how to create a DNA profile

Answer 12

Cut the DNA sample with an endonuclease
Place DNA sample in a well at the negative end of a gel
Turn on the electricity and the DNA fragments will move towards the positive end.
They will seperate based on size and charge.
Compare with standards which are of know size, that were also placed in the first well, to estimate the size of the other fragments.

Question 13

State the purpose of standards in an agarose gel

Answer 13

These are DNA fragments of known size (bp) that can be used to compare and then estimate the size of the other fragments.

Question 14

An endonuclease is

Answer 14

Bacterial enzyme that cuts DNA at a specific recognition sequence

Question 15

Gel electrophoresis sorts DNA based on

Answer 15

size and charge

Question 16

Gene cloning is

Answer 16

Making multiple copies of a gene, usually within bacteria in order to express the gene product

Question 17

A plasmid is a

Answer 17

Small ring of bacterial DNA can be used as a vector for DNA recombination and insertion

Question 18

Bacterial transformation is a process by which

Answer 18

a bacterial cell takes up a recombinant plasmid and expresses the genes of the plasmid

Question 19

Reverse transcriptase is a

Answer 19

Retrovirus enzyme that copies mRNA into copy DNA

Question 20

Define a GMO

Answer 20

An organism whose genome has been altered

Question 21

Define a TGO

Answer 21

Genetically modified organisms where genes from a different species are added to their genome

Question 22

In gel electrophoresis DNA moves from the ______ end to the ______ end.

Answer 22

Negative to positive end

Question 23

A DNA probe is

Answer 23

a single-strand segment of DNA which is (radioactively) labelled.

Question 24

How are bacteria that have been transformed identified?

Answer 24

Plasmid includes an antibiotic resistant gene. Bacteria are grown on agar containing that antiobiotic so that only those that are transformed will survive

Question 25

Genetic screening is …

Answer 25

testing an individual to identify the presence of a particular allele.

Question 26

Steps in gene cloning are …

Answer 26

Use reverse transcriptase to get c.DNA of the gene of interest
Cut gene and plasmid using the same endonuclease.
Stick gene into plasmid using DNA ligase which joins the sugar phosphate backbone.
Transform bacteria using heat or electrical shock
Check for successful transformation (usually using an antibiotic or GFP). Untransformed bacteria will die with the antibiotic. Successful will express the gene making the protein.

Question 27

Steps to modify plants using agrobacteria include

Answer 27

Identify gene that codes for the protein of interest and cut this out using an endonuclease. Cut a plasmid. Join the gene of interest to the plasmid using DNA ligase. Transform Bacteria (Agrobacteria) with the plasmid. Bacteria will then enter the plant’s cells and then inject the plasmid DNA into the host genome. This will then be able to be expressed.

Question 28

CRISPR stands for

Answer 28

clustered regularly interspaced short palindromic repeats.

Question 29

CRISPR Cas 9 was originally discovered in ..

Answer 29

Bacteria

Question 30

Describe the role of CRISPR in bacteria.

Answer 30

CRISPR is the bacteria’s memory of previously encountered viral DNA stored as spacers. This means that it is a primitive adaptive immune system.

Question 31

What are the two components of using CRISPR technology in other organisms?

Answer 31

Cas 9 enzyme and a single guide RNA ( sgRNA)

Question 32

Explain the process of attenuation, when there is low levels of tryptophan present.

Answer 32

Because tryptophan is present, the ribosome runs past the tryptophan codons and stops at the stop codon between domains 1 and 2.

This then prevents 2 from pairing with 3, leaving 3 free to pair with 4, forming a ‘hairpin’.

Therefore putting tension on the attenuator, so the mRNA pulls away from the DNA, causing RNA polymerase to fly off and ending transcription of the structural genes.•

Question 33

Explain the process of attenuation, when there is no tryptophan present.

Answer 33

Because tryptophan is absent, the ribosome pauses at the two tryptophan codons, waiting for a tRNA carrying tryptophan.

Then Domain 1 is covered and allows Domain 2 to pair with 3, preventing Domain 3 pairing with 4.

Therefore this creates a hairpin, but because it’s a fair way from the attenuator, the mRNA does not pull away from the DNA, and the RNA polymerase continues to slide along, transcribing the structural genes of the operon (TrpE - TrpA).

Question 34

State enzymes used in gel electrophoresis

Answer 34

Endonuclease

Question 35

State enzyme used in PCR

Answer 35

DNA Taq polymerase

Question 36

Describe the steps in using CRISPR technology to edit genomes in other organisms

Answer 36

1. Identify the nucleotide sequence of the __________ target gene.
2. Make single guide RNA which is complementary to the _______ target DNA.
3. This is joined with Cas9 to form the sg.RNA Cas9 complex.
4. Sg.RNA guides Cas9 to the target DNA and Cas9 cuts up the DNA.
5. The cell will then attempt to repair the sequence. Sometimes a corrected DNA sequence is also put into a target cell to be inserted after the target gene is cut.

Question 37

State what viral DNA is stored as in bacteria as part of CRISPR.

Answer 37

Spacers

Question 38

Describe the function of PAM in a prokaryote

Answer 38

PAM allows an attachment site for Cas9 on the bacteriophage DNA, not in the CRISPR gene locus.

This means that the g.RNA Cas9 complex will only attach to the specific and complementary viral DNA when PAM matches and not attach and cut up the viral DNA stored as spacers as part of CRISPR.