DNA manipulation

Question 1

restriction endonuclease

Answer 1

endonucleases that target a specific recognition site (restriction site)

Question 2

DNA ligase

Answer 2

enzymes that anneals 2 fragments of DNA.
- form phosphodiester bonds

Question 3

DNA polymerase

Answer 3

add nucleotides to DNA
- require a primer

Question 4

taq polymerase

Answer 4

a heat resistant polymerase required to bind new nucleotides to single stranded DNA
-ideal temp: 72 degrees celcius

Question 5

blunt ends

Answer 5

(restriction enzymes create) a straight cut through DNA with no overhanging nucleotides

Question 6

sticky ends

Answer 6

(restriction enzymes create) a staggered cut through DNA which results in overhanging nucleotides

Question 7

CRISPR in bacteria

Answer 7

• primative adaptive immune system
• defence mechanism
• used to protect against bactertiophage infection by cutting viral DNA preventing them from replicating

Question 8

PAM (protospacer adjacent motif)

Answer 8

short sequence of nucleotides that Cas1 and Cas2 recognise.
they are signalled to extract the protospacer by cutting viral DNA just before the PAM.

Question 9

Cas1 & Cas2

Answer 9

run along viral DNA looking for a PAM sequence. cut out a short section of DNA called the protospacer, just before the PAM sequence.
(CRISPR associated enzymes)

Question 10

protospacer

Answer 10

section of viral DNA cut out by Cas1 and Cas2 before being inserted into the CRISPR gene

Question 11

spacer

Answer 11

short sequences of viral DNA that are inserted into the CRISPR sequence

Question 12

repeat

Answer 12

palindromic repeats of DNA sequences in between spacers in the CRISPR array

Question 13

CRISPR array

Answer 13

contains repeats and spacers. part of the CRISPR array is (spacer and part of repeat) is turned into gRNA

Question 14

gRNA (guide RNA)

Answer 14

CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it.
specific sequence determined by CRISPR to guide Cas9 to a specific site.

Question 15

sgRNA (single guide RNA)

Answer 15

guide RNA created by scientists.
to instruct Cas9 to cut a specific site (when using CRISPR-Cas9 in gene editing)

Question 16

Cas9

Answer 16

endonuclease that creates a blunt cut at a site specified by gRNA

Question 17

CRISPR-Cas9

Answer 17

a complex formed between gRNA and Cas9 which can cut a target sequence of DNA.
bacteria defence mechanism against viruses
used fro genome editing by scientists

Question 18

why is DNA altered after Cas9

Answer 18

when the DNA is cut, enzymes will try to repair it.
repair mechanisms are prone to errors so Cas9 will continue to cut that target DNA until a mutation occurs. the gene is now non-functional.

Question 19

gene knock in

Answer 19

to add DNA segments.
scientists add them to the cell in hope they will be used in the post-cut repair process.

Question 20

gene knock out

Answer 20

scientists prevent the expression of a target gene to understand its funtion in an organism

Question 21

PCR (polymerase chain reaction)

Answer 21

used to amplify DNA by making identical copies

Question 22

purpose of PCR

Answer 22

used whenever there I’d an insufficient DNA sample for testing
- analysing gene fragments for genetic testing
- paternity testing
- forensic testing

Question 23

materials required for PCR

Answer 23

• DNA sample
• taq polymerase
• source of nucleotides
• DNA primers
  (all placed in a thermocycler)

Question 24

PCR stage 1

Answer 24

Denaturation:
- DNA sample is heated to 90-95 degrees celcius
-hydrogen bonds break to form single stranded DNA

Question 25

PCR stage 2

Answer 25

annealing: - single stranded DNA is cooled to 50-55 degrees celcius - allows primers to bind

Question 26

PCR stage 3

Answer 26

elongation: - DNA is heated to *exactly* 72 degrees celcius, optimal temp. of taq polymerase - taq polymerase binds to the primers and synthesises new strand of DNA (cycle is repeated 35-45 times)

Question 27

purpose of primers in PCR

Answer 27

forward primer: binds to the start codon at the 3' end of the template strand reverse primer: binds to the stop codon on the 3' end of the coding strand Allows taq polymerase to synthesise new DNA strand.

Question 28

applications of gel electrophoresis

Answer 28

used to measure the size of DNA fragments. - can be used to detect genetic disorders like cystic fibrosis - can be used for DNA profiling by comparing STRs

Question 29

factors affecting how a DNA fragment will run

Answer 29

- voltage - gel composition - buffer concentration - time

Question 30

STRs (short tandem repeats)

Answer 30

small sections of repeated nucleotides that vary in length between people

Question 31

gel electrophoresis steps

Answer 31

1. DNA samples are placed in wells at one end of the gel 2. an electric current is passed through the gel 3. smaller fragments of DNA move faster/travel further through the gel 4. DNA is stained to make bands visible

Question 32

why is a standard ladder used when reading gels

Answer 32

standard ladder contains DNA of a known length. it is compared to the DNA samples being analysed to estimate their length

Question 33

plasmid

Answer 33

small circular loop of DNA separate from the chromosome. typically found in bacteria.

Question 34

recombinant plasmid

Answer 34

circular DNA that is ligated to incorporate a gene of interest

Question 35

bacterial tranformation

Answer 35

the process by which bacteria take up foreign DNA from their environment. used to introduce recombinant plasmids into bacteria

Question 36

reporter gene

Answer 36

gene with an easily indentifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest

Question 37

electroporation

Answer 37

delivering an electric shock to bacterial membranes to increase membrane permeability, and increase the likelihood of bacterial transformation

Question 38

heat shock

Answer 38

bacteria and plasmids are placed in a calcium ion solution on ice. it is then rapidly heated and cooled to increase membrane permeability, and increase the likelihood of bacteria transformation

Question 39

lacZ enzyme

Answer 39

- lacZ gets inserted into the recombinant plasmid, already containing the insulin subunit gene. - lacZ produces beta-galactosidase

Question 40

beta-galactosidase

Answer 40

- creates a fusion protein with the insulin subunit protein which protects it from being digested by the cell. - identifiable marker because it turns x-gal blue when plated with it. *signals it has taken up the recombinant plasmid*

Question 41

how insulin is isolated from bacteria

Answer 41

- cyanogen bromide solution is added to bacteria - breaks down methionine at the start of the insulin gene, separating it from the beta galactosidase - can now be isolated and purified

Question 42

genetic modification

Answer 42

the manipulation of an organisms genetic material using biotechnology

Question 43

cisgenesis

Answer 43

process of inserting DNA from the same species

Question 44

transgenesis

Answer 44

process of inserting DNA from another species

Question 45

how a transgenic plant is made

Answer 45

1. desired trait is identified and isolated 2. gene of interest is inserted into the plant cell 3. transformed plant cell is grown repeatedly in a lab before being transferred to fields