03. 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 (e.g. human insulin mRNA) into c.DNA so that it contains no introns.

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

Outline the steps taken to produce human insulin from bacteria.

Answer 24

Isolate gene for insulin chain A
Use reverse transcriptase to get c.DNA from isolated mRNA so that it contains no introns.
Cut gene and plasmid using the same endonuclease.
Stick insulin gene into plasmid using DNA ligase which joins the sugar phosphate backbone.
Transform bacteria using heat or electroporation and check for successful transformation by growing on an antibiotic plate with ampicillin and then an antibiotic plate with tetracycline
Bacteria with no plasmid will not grow on the ampicillin plate, successfully transformed bacteria with the non-recombinant plasmid will grow on both plates and the successfully transformed bacteria with the recombinant plasmid will only grow on the ampicillin plate.
Purify plasmids, cut with an endonuclease and stick β-galactosidase gene into plasmid using DNA ligase
Transform bacteria using heat or electroporation and check for successful transformation by growing on X-gal agar plate
Bacteria with no β-galactosidase gene will appear white and bacteria with the β-galactosidase gene will appear blue, collect blue colonies
Repeat for insulin chain B
Purify insulin chains A and B and join them together to create a functional insulin

Question 25

CRISPR Cas 9 was originally discovered in ..

Answer 25

Bacteria

Question 26

Describe the role of CRISPR in bacteria.

Answer 26

Primitive adaptive immune system this means that
Invading viral DNA is cut and stored as spacers in the CRISPR array providing a memory of viral infections
If the same virus reinfects the bacteria, gRNA is transcribed and attached to a Cas9 endonuclease forming a gRNA-Cas9 complex
gRNA guides Cas9 to the viral DNA and Cas9 recognises the PAM sequence and then cuts the viral DNA, preventing destruction of the bacteria.

Question 27

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

Answer 27

Cas 9 enzyme and a single guide RNA ( sgRNA)

Question 28

State enzymes used in gel electrophoresis

Answer 28

Endonuclease

Question 29

State enzyme used in PCR

Answer 29

DNA Taq polymerase

Question 30

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

Answer 30

Identify the nucleotide sequence of the ______ target gene.
Make single guide RNA which is complementary to the ____ target DNA.
This is joined with Cas9 to form the sg.RNA Cas9 complex.
Sg.RNA guides Cas9 to the _____ gene and Cas9 cuts the DNA.
Transcription/no transcription of the _____ gene will occur and a functional ____ protein/non-functional _____protein is produced.

Question 31

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

Answer 31

Spacers

Question 32

Describe the function of PAM in a prokaryote

Answer 32

Acts as the binding site for the cas9 endonuclease and allows cas9 to cut the DNA sequence.
The PAM sequence plays an essential role in distinguishing self (CRISPR) from non-self (viral DNA).
Allows for faster recognition of viral DNA when it enters a cell

Question 33

Use the explain scaffold to justify an organism as being a genetically modified organism.

Answer 33

Because a genetically modified organism is one whose genome has been altered whereas a transgenic organism has had a gene from a different species added to its genome
Then as the ____ has had its genome altered without the addition of a different species gene
It is therefore a genetically modified organism

Question 34

Use the explain scaffold to justify an organism as being a genetically modified organism and transgenic organism.

Answer 34

Because a genetically modified organism is one whose genome has been altered whereas a transgenic organism has had a gene from a different species added to its genome
Then as the ____ has had its genome altered with the addition of a different species (_______) gene
It is therefore a genetically modified organism and transgenic organism.