Chapter 4

Question 1

. Endonucleases

Answer 1

Enzymes that cleave DNA

Question 2

Restriction endonucleases

Answer 2

Enzymes that cleave DNA at the corresponding site

Question 3

Blunt ends

Answer 3

Straight cut, no overhanging nucleotides

Question 4

Sticky ends

Answer 4

Staggered cut, overhanging nucleotides

Question 5

Ligases

Answer 5

Enzymes that join DNA and form phosphodiester bonds to merge

Question 6

Polymerase

Answer 6

Synthesis of DNA molecules

Question 7

Probe

Answer 7

Used to locate specific DNA sequences

Question 8

Cas-9

Answer 8

Endonuclease that cleaves DNA according to the PAM sequence
Molecular scissors

Question 9

PCR

Answer 9

Amplify DNA

Question 10

Gel electrophoresis

Answer 10

Tool used to identify the length of unknown DNA

Question 11

Gene of interest

Answer 11

Gene to be inserted into a genome

Question 12

Plasmid

Answer 12

Small circular pieces of DNA found in bacteria

Question 13

What does CRISPR stand for

Answer 13

C- clustered
r- regularly
I - interspaced
s-short
P- palindromic
R- repeats

Protect prokaryotes from viral invasion

Question 14

Bacteriophage

Answer 14

A virus that infects prokaryotic organisms

Question 15

CRISPR - Cas 9

Answer 15

A complex formed between gRNA and Cas 9 which can cut a target sequence of DNA. Bacteria use this complex for protection from viruses

Question 16

gRNA

Answer 16

Guide rna

Question 17

PAM

Answer 17

site for cas 9 to cleave dna

Question 18

Spacer

Answer 18

Viral dna stored in the bacterial genome

Question 19

Protospacer

Answer 19

Viral dna before spacer

Question 20

Steps of CRISPR CAs 9

Answer 20

1. Exposure
2. Expression
3. Extermination

Question 21

Exposure

Answer 21

Bacteriophage injects its dna into a bacterium which identifies the viral dna as a foreign substance
Enzymes cut out protospacers from viral DNA
Protospacers introduced into bacterium’s CRISPR gene

Question 22

Expression

Answer 22

gRNA is produced that matches target dna
gRNA binds to CAs 9 to create CRISPR cas9 complex

Question 23

Extermination

Answer 23

CRISPR CAs 9 complex finds mutated dna that is complementary to gRNA
CAs 9 cuts out mutated sequence
Double stranded break in target dna
Faulty gene can be replaced by corrected or new gene
Cells attempt to repair the break effectively silences the target gene

Question 24

PCR - polymerase chain reaction

Answer 24

DNA manipulation technique that amplifies dna by making multiple copies
DNA polymerase is known as taq dna polymerase

Question 25

1. Denaturation

Answer 25

DNA heated to 90-95 to break the hydrogen bonds between nucleotides and seperate the strands of dna

Question 26

2. Annealing

Answer 26

Single stranded dna is cooled to approx 50-55 to allow the primer to bind to complementary sequences on the single stranded dna

Question 27

3. Elongation

Answer 27

DNA is heated to 72 which allows taq dna to work optimally. Binds to primer and begin synthesising a complementary strand of dna using free floating nucleotides

Question 28

4. Repeat

Answer 28

Create more copies

Question 29

Forward primer

Answer 29

Binds to start codon 3’. Template strand Taq dna synthesis dna in the same direction that rna polymerase would

Question 30

Reverse primer

Answer 30

Binds to stop codon 3’. Coding strand taq dna to synthesise a new dna strand in the reverse direction that rna polymerase would

Question 31

Gel electrophoresis

Answer 31

A lab technique to measure the size of dna fragments Seperate dna based on size 1. DNA loaded into wells at negative end. Standard ladder 2. Electric current passed through for a particular time 3. DNA separates. smaller fragments move faster therefore down 4.dna stained with dye. Visible under uv light

Question 32

Factors of gel electrophoresis

Answer 32

Timer Voltage Buffer Concentration of gel

Question 33

STR - short tandem repeats

Answer 33

Small sections of repeated nucleotides found in introns It 2 samples match then u assume its the same persin

Question 34

Plasmid vectors

Answer 34

Plasmid is an autonomously replicating circular dna found in bacteria and yeast

Question 35

Recombination and transformation

Answer 35

Isolated plasmid vector. Restriction site is cut Gene of interest is removed DNA ligase. = recombinant plasmid Binary fission Heat shock or electroporation

Question 36

Heat shock

Answer 36

Calcium chloride and different temps to enable uptake of the plasmid dna

Question 37

Electroporation

Answer 37

Electrical current passes through solution with bacteria and plasmid vectors to make it more permeable

Question 38

Antibiotic selection

Answer 38

To distinguish between transformed and untransformed bacteria, the mixture is cultured onto an antibiotic-rich plate. All untransformed will die

Question 39

Insulin

Answer 39

Alters the body’s ability to regulate blood glucose

Question 40

Recombinant insulin

Answer 40

Different insulin types depending on the symptoms that need to be treated 1. Creating the recombinant plasmid 2. Creating transformed bacteria 3. Protein production and extraction

Question 41

Insulin clone

Answer 41

Make dna that encodes the pre-pro insulin by PCR Include a Restriction site on either side of dna fragment Choose a vector that has the restriction site DNA ligase to join sticky ends Puc18 uses a blue/white screening system that involves lacz Large scale fermentation to mass produce the protein

Question 42

GMO - genetically modified organisms

Answer 42

Gene modified in some way Adding a gene, silencing gene, changing regulatory gene

Question 43

Transgenic

Answer 43

Animal or plant that has a gene which has never existed in its genetic history

Question 44

Ethical issues of genetic engineering

Answer 44

Approval Ownership of technology Impact on native species What happens when things go wrong