Recombinant DNA technology

Question 1

What is recombinant DNA?

Answer 1

The introduction of a foreign gene into the DNA of another organism. The resulting organism contains DNA from 2 types of organisms

Question 2

With the introduction of recombinant DNA what is the resulting organism known as?

Answer 2

A genetically modified organism (GMO)

Question 3

What is the process of making a protein using DNA technology/ gene transfer and cloning?

Answer 3

1. Insolation of DNA fragment that have the gene for the desired protein
2. Insertion- insertion of DNA fragment into a vector
3. Transformation- Introduction of DNA fragment into suitable host cell
4. Identification- Identification of host cells that have taken up the DNA using gene markers
5. Growth/ cloning- Culturing of host cells containing the DNA to produce the protein on a large scale

Question 4

Why does a DNA fragment from one organism, inserted into another, still produce the same protein?

Answer 4

It is universal- the same codon codes for the same amino acids

Question 5

What is the first method of the isolation of DNA fragments?

Answer 5

Using reverse transcriptase an enzyme that works backwards to produce DNA from mRNA
1. The mRNA has been transcribed from the gene of interest
2. Reverse transcriptase is used to synthesise a single strand of complimentary DNA (cDNA) from the mRNA molecule
3. DNA polymerase then forms the other strand of DNA from free nucleotides > double strand

Question 6

What are the advantages of using mRNA rather than DNA?

Answer 6

Quantity only one copy of DNA per cell whilst gene may have been transcribed many times so more copies of mRNA available.
Searching- mRNA separate to DNA, easier to find and only ome DNA is transcribed in each cell (smaller genetic material to search through)
Genetic code- no introns

Question 7

What is the 2nd method for the isolation of DNA fragments?

Answer 7

Restriction endonucleases are enzymes found in bacteria that cut DNA at specific base (recognition sequences) so can be used to cut out a desired gene from the rest of the genome. Different restriction enzymes cut genes in many different ways

Question 8

What is the difference between blunt and sticky ends?

Answer 8

B- B- A- G- C- T- B- B
/ / / / > / / / /
B- B- T- C- G- A- B- B
|________________|
Blunt ends

-B -B -G A- A- T- T- C- B- B
/ / / > / / /
-B -B -C -T -T -A -A G- B- B
|_________________|
Sticky ends

Question 9

What are cut ends?

Answer 9

Are cut ends of DNA
One strand is longer than the other/ staggered cut
Can attach go complementary DNA bases
Will join with another sticky end but only if it has been cut with the same restriction endonuclease

Question 10

What are restriction endonucleases what is their function?

Answer 10

They have highly specific active site that catalyse the hydrolysis of the sugar phosphate backbone of both strands of the DNA molecule
Cut DNA at recognition sites between 4-8 base pairs long
Recognition sites are usually palindromic, meaning the sequence and its complement are the same but reversed
The recognition sequence is defined as a short specific palindromic base sequence

Question 11

What is the third method for the isolation of DNA fragments?

Answer 11

Fragments can also be produced by creating a gene in a ‘Gene machine in a lab
1. Desired nucleotide sequence derived from desired protein (protein > amino acid > mRNA > DNA
2. Nucleotide sequence fed into computer, checked and oligonucletides designed= small overlapping, single strands which assemble to form the complete gene
3. Oligonucleotides are then created and joined to make the gene
4. The gene is then replicated many times using PC, which also makes the gene into double stranded DNA
5.Using sticky ends the gene may be inserted into a bacterial plasmid (acts as a vector)

Question 12

What is a plasmid?

Answer 12

A circular piece of DNA separate from the main bacterial DNA and contains only a few genes

Question 13

What is the importance of sticky ends?

Answer 13

If DNA from different organisms is cut by the same restriction endonuclease and mixed, recombinant will be produced
Once the bases have paired up they form their usual weak hydrogen bonds between each other.The link between the sugar phosphate backbones is catalysed by the enzyme DNA ligase via a condensation reaction - ligation

Question 14

How do you prepare the DNA fragment for insertion?

Answer 14

Before being inserted, extra lengths of DNA must be added to the DNA fragment, these include:

A prometer- length of DNA added before the fragment, to which transcription factors and RNA polymerase can bind to initiate transcription
A terminator- Length of DNA added after the DNA fragment, which causes RNA polymerase to be released and stops transcription

Question 15

What is the second step in the process of protein synthesis using gene technology?

Answer 15

The insertion of genes into plasmids
Inserting our chosen gene into a plasmid, the plasmid acts as a carrier or vector. Which can then be introduced back into a bacterial cell

Question 16

What is the third step in the process of protein synthesis using gene technology?

Answer 16

Introducing our recombinant plasmids (vectors) into host cells
This is known as transformation
Plasmids are mixed with bacterial cells in an ice cold medium containing calcium ions
Heat shock (90° celcius) is applied for 2 mins. The bacterial cell membrane increases in permeability allowing the plasmids to pass through into the cell.
Only a few bacterial cells (0.01 percent) will take up the plasmids. So we need to identify which bacterial cells have been successful in taking up the plasmids

Question 17

What is the fourth step of the process of protein synthesis using gene technology?

Answer 17

Identification using gene markers
Three techniques can be employed that all involve using a second, separate gene on the plasmid (vector) which is easily identifiable

Question 18

What does the gene marker do?

Answer 18

Marker genes can be used to identify whether a gene has been taken up by bacterial cells
They all involve using a second, separate gene on the plasmid, this gene is easily identifiable as:
It may be resistant to an antibiotic, it may make a fluorescent protein that is easily seen
It may produce an enzyme whose action can be identified

Question 19

What are antibiotic resistance markers?

Answer 19

Some bacteria contain antibiotic resistance genes in their plasmids. A few bacteria have two genes for resistance to two different antibiotics in the same plasmid
Any bacterial cell possessing this plasmid would be resistant to both antibiotics (ampicillin and tetracycline)

Question 20

What is the method of finding out whether bacteria have taken up the desired plasmids using fluorescent markers?

Answer 20

Some organisms (jellyfish) produce a range of fluorescent proteins
These proteins must have their own genes which can be isolated and then introduced into bacterial cells (via plasmids)
1. Insert the gene for a fluorescent protein into the plasmid (vector) that also has the gene for ampicillin resistance
2. Insert your gene of interest (insulin) into the centre of a gene for a fluorescent protein
3. Transfer the plasmids into bacterial cells and grow in agar containing ampicillin
4. Any bacterial cells that successfully took up plasmids, will not be killed and will not be able to glow

Question 21

What is the method of finding out whether bacteria have taken up the desired plasmids using enzyme markers?

Answer 21

Involves inserting your gene of interest (insulin) into a gene that codes for the enzyme lactose
1. There is a particular substrate that is usually colourless, but turns blue when lactase acts apon it
2. Insert your chosen gene into the gene that makes lactase, thereby inacivating the lactase gene. This plasmid also contains the gene for ampicillin resistance
3. Transfer the plasmids into bacterial cells
4. Grow in agar containing bacterial cells and the colourless substrate
5. Any colonies that have taken up the plasmid will not be able to change its colour to blue
6. Any colourless spots will indicate to you, which cells have been transformed

Question 22

What is in vivo?

Answer 22

when it is performed in a living organism.

Question 23

What is in vitro?

Answer 23

Performed in a test tube /culture dish (outside a living organism)

Question 24

What is the polymerase chain reaction (PCR)?

Answer 24

Method of replicating DNA in the lab
create millions of copies DNA is a few hours

Question 25

What is the purpose of the polymerase chain reaction?

Answer 25

Small samples of DNA are difficult to analyse, so essential in forensic science
Amplifys DNA so that it can be analysed
Provide sufficient mass of DNA for genetic fingerprinting

Question 26

What is needed for the polymerase chain reaction?

Answer 26

1 . The DNA fragment to be copied
2. DNA polymerase - Joins DNA nuletides, obtained from not spings when I’ve- thermostable
3. Primers- short single stranded length of DNA. They have a base sequence complementary to start of section of DNA to be amplified. Allows attachment of DNA polymerase and prevents the two strands from rejoining.
4. Free DNA nucleotides- A,G,C,T

Question 27

what are the stages of the polymerase chain reaction?

Answer 27

1 . Strand separation DNA heated at 95 C for 5 mins
Mix with primers
2. Annealing for primers mixture cooled to 55 c with free nucleotides and thermostable DNA polymerase
3. DNA synthesis Mixture heated to 70°2
Optimum temp for enzyme
REPEAT CYCLING - with every cycle the amount of DNA increases two times

Question 28

What are the advantages of using recombinant DNA technology as opposed to selective breeding to improve the productivity of crops and animals?

Answer 28

It’s possible to alter the genotype of an organism by transferring genes between individuals of the same species