Y2: Gene technology

Question 1

What are the three methods to make/produce DNA fragments?

Answer 1

1. mRNA to make copy with reverse transcriptase
2. Restriction endonucleases
3. Gene machine

Question 2

What is the method for using reverse transcriptase to copy the insulin gene?

(Using reverse transcriptase to make DNA fragment (gene))

5

Answer 2

1. Beta cells from the islets of langerhans produce insulin mRNA
2. Reverse transcriptase uses DNA bases to make a single stranded complementary DNA (cDNA)
3. cDNA separated from mRNA by hydrolysis with an enzyme
4. DNA polymerase forms a second strand from the cDNA
5. This forms a full copy of the human insulin gene

Question 3

What is the method for using restriction endonucleases to make DNA fragments?
2
Blunt ends

Answer 3

1. Restriction endonuclease has a recognition site for the DNA sequence it cuts at this site is not palindrome
2. This produces a straight cut so blunt ends

Question 4

What are restriction endonucleases?

2

Answer 4

Enzymes that hydrolyse DNA

Cut DNA a known sequences

Question 5

What is the method for using restriction endonucelases to make DNA fragments?
2
Sticky ends

Answer 5

1. Restriction endonuclease has recognition site for DNA sequence that is in a palindrome sequence
2. Cuts and produces a staggered cut so makes sticky ends

Question 6

What are the two types of DNA fragment restriction endonucleases make?

Answer 6

Blunt ended

Sticky ended

Question 7

How are DNA fragments produced using the gene machine?

4
extra
1

Answer 7

1. Use bioinformatics to determine base sequence of gene
2. Computer designs series of oligonucleotides and they are assembled
3. The oligonucleotides are then assembled to form a gene
4. Replicated by polymerase chain reaction
   (5. Using sticky ends it can inserted into plasmid (vector))

Question 8

What are the advantages of using the gene machine?

3

Answer 8

1. Any sequence can be produced in a short time
2. Has great accuracy
3. Free of introns so can be transcribed and translated by prokaryotic cells

Question 9

What are the advantages of in vitro gene cloning?

2

Answer 9

1. Extremely rapid so is valuable when there is only a small bit of DNA
2. Does not require living cells

Question 10

What is a disadvantages of in vitro gene cloning?

Answer 10

As it copies it rapidly if there is contamination that also multiplies by loads

Question 11

What is used in the polymerase chain reaction?

5

Answer 11

1. DNA fragment to be copied
2. DNA polymerase
3. Primers
4. Nucleotides
5. Thermocycler

Question 12

What is the method for the polymerase chain reaction?
1. Separation of the DNA strand

2

Answer 12

1. DNA fragments and primers are put into a vessel and into a thermocylcer to vary the temperature over time
2. Temperature is then increased to 95*C causing the H bonds between the DNA strands to break separating the DNA strands

Question 13

What is the method for the polymerase chain reaction?
2. Addition of primers

3

Answer 13

1. The mixture is cooled to 55*C causing the primers to join/anneal to their complementary bases at the end of the DNA fragment
2. Primers attach to provide starting sequences for DNA polymerase to begin copying from and prevents the strands from rejoining
3. DNA polymerase is added

Question 14

What is the method for the polymerase chain reaction?
3. Synthesis of DNA

3

Answer 14

1. Temperature is cooled to 72*C, this is the optimum temperature for DNA polymerase
2. DNA polymerase adds nucleotides along the separated DNA strands beginning at primer and ending at end
3. It catalyses the formation of phosphodiester bonds

Question 15

What does in vitro mean?

Answer 15

Using test tube/scientific equipment to make something

Question 16

What does in vivo mean?

Answer 16

In organism

Question 17

Why are sticky ends important?

3

Answer 17

1. The ends of the fragment have unpaired bases
2. The two ends are complementary to each other
3. DNA ligase forms phosphodiester bonds between nucleotides once complementary bases have lined up to create one piece of combinant DNA

Question 18

What are the issues with using sticky ends?

3

Answer 18

1. Plasmid sticky ends can stick to each other not the gene that you are trying to insert
2. Sticky ends of the gene can stick to each other creating a loop not associated with the plasmid
3. RNA polymerase and transcriptional factors need promoter region on DNA to recognise where to start transcription

Question 19

In Vivo Cloning? How is the DNA inserted into the host cell by a vector?

4

Answer 19

1. Use a vector eg: plasmid
2. Plasmids usually carry genes for antibiotic resistance (you need to know which ones)
3. Cut the plasmid with the same restriction endonuclease as to make the DNA fragments so you get complementary ends
4. Mix the fragments with the cut plasmids and DNA ligase

Question 20

In Vivo Cloning: How are the plasmids inserted into the bacteria?

3

Answer 20

1. Mix the plasmids and the bacteria in a medium containing Ca2+
2. Modify the temperature
3. Some bacteria will have taken up the plasmids and some of the plasmids will have the incorporated gene (transgemic plasmids)

Question 21

In Vivo Cloning: how do you identify which plasmids have taken up the gene?

method 1
2
method 2
2

Answer 21

1. Fluorescent markers: insert gene for fluorescence into plasmid,
2. 5. If it is fluorescent there is no gene as the gene is inserted into the fluorescence gene and it is still functional
3. Enzyme markers: lactase as it turns colourless substrate blue
4. 5. I f it is blue there is no gene as the lactase is still functional so gene is not inserted

Question 22

How does DNA hybridisation work? (DNA probes)

5

Answer 22

1. Take a sample of the DNA to be tested
2. Heat it up to 95*C to break H bonds
3. Mix sample and DNA probe together
4. Cool it down so the probe can attach to the sample DNA - allows H bonds to form
5. Detect to see if probe has bound to DNA
   - Wash off solution from fixed surface
   - Detect

Question 23

What is a DNA probe?

2

Answer 23

Short, single stranded length of DNA with a label attached

-Can be a fluorescent molecule or radioactive nucleotide

Question 24

What are the advantages of genetic screening?

2

Answer 24

You can test for specific alleles so:
1. can do personalised medicine
2. genetic counselling with experts to discuss risks and decisions
Eg: about having children or treatments etc.

Question 25

How do DNA probes work? Method for using them for genetic screening?

8

Answer 25

1. Use genome library to get the sequence for the allele for testing
2. Make complementary probe of allele of interest
3. Copy probe using PCR
4. Label all probes
5. Put lots of different probes on one slide (assay)
6. Apply sample
7. Wash off solution, probes that are unbound wash off
8. Detect probes that are bound (film etc and line up with probe map)

Question 26

What are variable number tandem repeats (VNTR’s) and how does genetic fingerprinting rely on them?
2

Answer 26

1. Repetitive non-coding DNA bases

2. Number and length is unique to a person so you can differentiate between different people

Question 27

What are the stages of genetic fingerprinting? Short

5

Answer 27

1. Extraction
2. Digestion
3. Separation
4. Hybridisation
5. Development

Question 28

What are the stages of genetic fingerprinting long version?

6

Answer 28

1. Extract sample of DNA and amplify w/ PCR
2. Cut DNA sample into fragments using restriction endonucleases (different lengths for different people)
3. Use gel electrophoresis to separate the fragments
4. Put in alkali solution to separate the strands and bind to a nylon membrane
5. Bind DNA markers to VNTR’s
6. Use x-ray film etc. over the membrane to get pattern of VNTR’s

Question 29

What are the uses of genetic fingerprinting?

4

Answer 29

1. Genetic relationships: parentage, genetic diversity
2. Forensic science
3. Medical diagnosis: number of repeats alters severity of illness
4. Plant and animal breeding: pedigree, prevent undesirable breeding etc.