Genetic Engineering

Question 0

What way can genetic material be modified?

Answer 0

Insert foreign gene from 1 organism to another= transgenic
Altering an existing gene so that it’s product is changed
Changing a gene expression so that it’s translated more often or not al all.

Question 1

What is genetic engineering?

Answer 1

Altering the genes in a living organism to produce a genetically modified organism with a new genotype.

Question 2

What are restriction enzymes?

Answer 2

They cleave the DNA apart.
2 types
1. Endonuclease- cleave within DNA
2. Exonuclease -cleave at end of DNA

Question 3

How are the 4 types of endonuclease classified?

Answer 3

Subunit composition
Cleaveage position
Sequence specify
Co factor requirement

Question 4

What is the main endonuclease used in gene cloning?

Answer 4

Type 2

Question 5

How do we isolate the gene?

Answer 5

Using restriction enzymes.
Cut at specific sites
Some cut straight across - blunt ends
Some make a staggered cut- sticky ends

Question 6

What happens to the sticky ends?

Answer 6

These sticky ends anneal to another piece of DNA by comp base pairing but only if they have been cut by the same restriction enzyme.

Question 7

What is PCR?

Answer 7

Polyemerase chain reaction. Used to amplify a number of copies of a specific region of DNA. Produce a large enough sample to be analysed
Eg. Forensic evidence or cell from a dinosaur

Question 8

What do you need for PCR?

Answer 8

A length of DNA mixed with 4 nucleotides. Add in taq polyemerase and the DNA will replicate many times under correct temp.

Question 9

List the steps in the PCR?

Answer 9

1. Denaturing. Heat break H bonds - DNA unzips
2. Annealing. Primers added and bind to ends of single strands
3. Extension step. DNA polyemerase uses free nucleotides to create complementary strands
4. Repeat cycle many times to amplify large amounts of dna.

Question 10

What are primers?

Answer 10

Short sequences that dna polyemerase recognise as start tags
You must know the nucleotide sequence just before and after the gene to be copied
Complementary forward and reverse primers are then created

Question 11

What temps do the PCR steps have be?

Answer 11

Denaturing and breaking H bonds: 95 degrees
Annealing: 50-60 degrees
Extension: 72 degrees

Question 12

What is gel electrophoresis?

Answer 12

Technique which separates DNA or proteins using electrical current.
Allows scientists to identify someone’s DNa
Separates pieces of DNA or proteins based on length.

Question 13

How does electrophoresis work?

Answer 13

DNA sample placed into wells at one end of a thin slab of gel made of agarose ( or polyacrylamide) and covered in buffer. Electric current passed through the gel. Negative charged DNA attracted to anode(+)
DNA diffuses through the tiny holes of agarose. Smaller length faster and further they move. At end of run current is turned off.

Question 14

How can DNA be visualised after electrophoresis?

Answer 14

Add ethiduim bromide- fluoresces under uv light. It’s carcinogenic.

Question 15

What steps are involved in genetic fingerprinting?

Answer 15

1. Cut DNA with restriction enzymes 2. Run DNA fragments through a gel. 3. Bands will form in the gel 4. Everyone’s DNA bands are unique.5. DNA bands are like genetic fingerprints.

Question 16

What processes are involved in DNA technology?

Answer 16

Isolation
Insertion
Transformation
Identification
Growth/ cloning

Question 17

How do you produce DNA fragments?

Answer 17

Retrovirus contain reverse transcriptase- turn viral RNA into DNA so it can be transcribed by host cell into proteins.
Makes DNA from RNA template
Example B cells in pancreas

Question 18

How is reverse transcriptase used to make insulin?

Answer 18

Extract mature mRNA coding for insulin
Single strand comp cDNA is formed using reverse transcriptase on mRNA template
Single stranded cDNA used to form double strand DNA using DNA polyemerase

Question 19

Why do bacteria contain restriction enzymes?

Answer 19

To protect themselves from invading viruses
Restriction enzymes used by bacteria to cut viral DNA
Cut at specific sites- “blunt” straight through “Sticky” staggered cut and will join another sticky end but only if cut by same rest. enzyme
Also called restriction endonuclease

Question 20

List the features of a restriction endonuclease ?

Answer 20

Highly specific active sites
Cut about 4-8 base pairs long- called recognition sites
Palindromic- the sequence and it’s compliment are the same but reversed

Question 21

Why are sticky ends important?

Answer 21

DNA from diff sources can be joined if they have same sticky ends- same recognition site
For this they must be cut with same endonuclease
Sticky ends joined by DNA ligase to join the sugar phos backbone
New DNA = recombinant DNA

Question 22

Why are plasmids useful as vectors?

Answer 22

Nearly always contain antibiotic resistance genes
1 of the antibiotic resistant genes is disrupted when restrict enzyme cuts open plasmid
Other antibiotic resist gene used in selection of the correct host cells

Question 23

What combinations of plasmids will occur during insertion of gene?

Answer 23

1. Hybrid- gene inserted and permanently bonded ( IDEAL)
2. Original plasmid- no change
3. Gene- circle of DNA sealed together ( no plasmid involved)
   All 3 are produced but only 1st is beneficial

Question 24

What happens during Transformation?

Answer 24

Plasmids reintroduced into host eg. Bacteria
This process= transformation
The bacteria, plasmids and calcium are mixed together
Altering temp the bacteria become permeable and plasmid can pass through the cell membrane

Question 25

What happens during the identification stage?

Answer 25

Only 0.001% bacteria take up DNA/PLasmids
Need to identify bacteria containing the plasmid- grow bacteria on medium containing antibiotic
Antibiotic resist gene found in plasmid therefore bacteria that survive must contain plasmid

Question 26

During the identification stage gene markers are needed to identify bacteria that have taken up the plasmid. What are the 3 gene markers used?

Answer 26

1. AntiB resist markers- see next question
2. Fluorescent markers- Green Fluorescent Protein (GFP) from jelly fish-DNA inserted into GFP gene bacteria will not glow and are identified. DNA not inserted into GFP bacteria will glow and not used
3. Enzyme markers- lactase turns colourless substance into blue

Question 27

How do antibiotic resistance markers work?

Answer 27

Ampicillin resist gene disrupted when restrict enzyme cuts plasmid
2nd antibiotic resist gene (resist to amp) identifies plasmid with DNA
If DNA has been inserted into amp resist gene it will stop growing on medium containing amp.
To identify these bacteria must use replica plating

Question 28

What does cloning involve?

Answer 28

After successful identification of bacteria contains plasmid and the DNA fragment - bacteria are cloned
As bacteria are cloned so is the plasmid containing the DNA fragment
This gene cloning is in viva ( inside organism)

Question 29

What are DNA probes?

Answer 29

A short length of DNA with a label attached

Used to identify and label DNA fragments that contain a specific sequence.

Question 30

What are the 2 types of gene probe?

Answer 30

Radio actively labelled probe- use photographic film
Fluorescently labelled probe
Probes are always single stranded DNA/RNA so they can anneal with base pair with comp sequence

Question 31

What use do DNA probes have?

Answer 31

Identify restriction fragments out of thousands formed from genomic library
Fingerprinting
Identify gene from 1 species that are similar to another species
Identify genetic defects