Genomes

Question 1

What is gel electrophoresis? (2 points).

Answer 1

1. Used to separate DNA fragments based on their size so they can be identified and analysed.
2. It is based on the fact that the rate at which a particular fragment of DNA moves is proportional to its mass.

Question 2

Describe the process of gel electrophoresis (3 points).

Answer 2

1. DNA is hydrolysed by restriction enzymes before it can be separated. The hydrolysed DNA sample is placed within the well at one end of the agarose gel. The gel is placed in a buffer so that the negative electrode is next to the well and an electrical current is passed through the gel.
2. As DNA is negtively charged (due to the phophate group), DNA fragments migrate toward the positively charged end of the gel / electrode.
3. Smaller DNA fragments migrate through the gel more quickly than larger fragements. So smaller fragments move further along the gel in the same period, this process separates fragments according to their size across the gel.

Question 3

Describe how the DNA fragments are visualised using Southern blotting and autoradiography (4 points).

Answer 3

1. An alkali is added to make the DNA single-stranded.
2. The DNA is transferred to a nylon membrane by Southern blotting.
3. Radioactively / fluorescently labelled DNA probes with complementary base sequences to the DNA and bind due to hydrogen bonding.
4. The DNA can be visualised using autoradiography (the radioactively labelled probes expose X-ray film so bands can be visualised).

Question 4

Describe DNA sequencing (16 points).

Answer 4

1. DNA is broken into fragments 200-600 base pairs long.
2. Adenine A-base is added to 3 prime ends of each fragment.
3. Adapters (short sequences of DNA) are attached to each end of the fragments.
4. Double strands of DNA are then separated into single strands.
5. These are washed across a flow cell (small plastic slide with primers- lots of small peices of DNA attached).
6. DNA bind to complementary primers.
7. Excess DNA is washed away.
8. DNA polymerase and bases are added.
9. A complementary strand of DNA is made.
10. The attached DNA fragment is used to make lots of copies via bridge amplification.
11. Complementary strand bends and is made along the bridge.
12. DNA is denatured.
13. Primers, DNA polymerase and fluorescently labelled DNA bases are added.
14. DNA polymerase adds fluorescent base.
15. Coloured lasers causes colours to grow.
16. Camera detects.

Question 5

Definition of proteome.

Answer 5

All the proteins produced in a given type of cell or organism at a given time.

Question 6

Definition of PCR.

Answer 6

To amplify fragments of DNA in an automated process.

Question 7

Definition of in vitro.

Answer 7

Experiment / processes carried outside living cells in test tubes.

Question 8

Definition of primers.

Answer 8

Short sequence of nucleotides that have a complementary base sequence to one end of the DNA strand to be copied. They allow the DNA polymerase to attach and start addition of nucleotides. They prevent strand re-joining.

Question 9

Describe polymerase chain reaction method (PCR)- an in vitro method of cloning (5 points).

Answer 9

1. Separation of DNA strand: the mixture is heated to 95 degrees which breaks the hydrogen bonds between bases, therefore separates the strands and produces single-stranded DNA molecules. DNA has become denatured.
2. Addition of the primers: the temperature is reduced to 55-60 degrees to allow primers to bind to the ends of the single-stranded DNA molecules- annealing.
3. Synthesis of DNA: the temperature is increased to 72 degrees. This enables the free DNA nucleotides to attach by complementary base pairing. The new DNA strands are built up using the enzyme tag polymerase which joins the phosphodiester bonds- extension. The first cycle is complete.
4. The cycle is repeated.
5. The 2 resulting DNA molecules make up the template DNA fro the next cycle, thus amplifying the amount of DNA duplicated for each new cycle.

Question 10

List the pros of in vivo cloning (7 points).

Answer 10

1. Very useful when introducing a gene into another organism, therefore can be used in gene therapy.
2. Uses vectors and plasmids which means the gene can be delivered into the other organism.
3. Produces transformed bacteria which can be used to produce large quantities of gene products, for example: insulin.
4. Almost no risk of contamination due to the use of restriction endonucleases and creation of sticky ends.
5. Very accurate, the copied DNA has very few errors as mutations are rare.
6. It can be used to copy genes which have not been studied before.
7. Reliably copies genes up to about 2 million base pairs long.

Question 11

List the cons of in vivo cloning (2 points).

Answer 11

1. It takes a long time to produce enough DNA.
2. Cannot be copiedunless isolated from other material.

Question 12

Pros of in vitro cloning (4 points).

Answer 12

1. Extremely rapid.
2. No valuable time is lost before forensic analysis and matchng can take place.
3. Does not require living cells, it can copy DNA which has been partly broken down
4. DNA embedded in other material can be copied.

Question 13

List the cons of in vitro cloning (5 points).

Answer 13

1. Any contaminating DNA found at a crime scene will massively be increased.
2. Requires a very pure sample to prevent contaminant DNA being multiplied.
3. An errors in copying DNA will also be copied in subsequent cycles.
4. It cannot be used to copy genes which have not been studeied before.
5. Unrelaible when copying fragments longer than 1000 base pairs long.

Question 14

Describe generally how to produce genetic fingerprints (8 points).

Answer 14

1. Extract the DNA: Crime scene / individual / cells and tissue.
2. Amplify the DNA: PCR.
3. Digestion of DNA: Restriction endonucleases.
4. Seaparation: Gel electrophoresis.
5. Southern blotting: Transfer to nylon sheet.
6. Hybridisation: Add radioactive / fluorescently labelled probes.
7. Development: Place nylon layer under UV light or expose to X-ray.
8. Interpreation: Make comparisons between bonds / locate specific genes using a DNA ladder.

Question 15

Describe how the technique for genetic fingerprinting is carried out. Markscheme 1 (10 points).

Answer 15

1. DNA is cut.
2. Using restriction enzymes.
3. Electrophoresis.
4. Separates according to length / mass / size.
5. DNA is made single-stranded.
6. Transfer to membrane / Southern blotting.
7. Apply probe.
8. Radioactive / single-stranded / detected on film / fluorescent.
9. Reference to tandem repeats / VNTR’s / minisatellites.
10. Pattern unique to every individual.

Question 16

Describe how the technique for genetic fingerprinting is carried out. Markscheme 2 (10 points).

Answer 16

1. DNA is extracted from sample.
2. DNA is cut / hydrolysed into segments using restriction endonucleases.
3. Must leave minisatellites / required core sequences intact.
4. DNA fragments separated using electrophoresis.
5. Detail of process, for example: mixture put into wells on gel and electric current is passed through.
6. Immerse gel in alkaline solution / two strands of DNA separated.
7. Southern blotting / cover with nylon / absorbent paper (to absorb DNA).
8. DNA fixed to nylon / membrane using UV light.
9. Radioactive marker / probe is added (which is picked up by required fragments) / complementary to minisatellites.
10. (Areas with probe) identified using X-ray film / autoradiography.

Question 17

Definition of VNTR’s.

Answer 17

Repetitive sequences of non-coding DNA.

Question 18

Write out the mark scheme perfect anser to describe how the technique of genetic fingerprinting is caarried out.

Answer 18

1. DNA is extracted from sample.
2. Amplify DNA using PCR.
   PCR:
   - DNA is heated to 90-95 degrees.
   - DNA strands separate as hydrogen bonds are broken between bases.
   - Cooled temperature to 55-60 degrees.
   - Primers bind (allow DNA polymerase to bind).
   - DNA nucleotides attach.
   - Complementary base pairing.
   - Temperature raised to 70-75 degrees.
   - Tag (DNA) polymerase joins nucleotides together.
   - Cycle is repeated.
3. DNA is cut / hydrolysed into segments using restruction endonucleases ; cut DNA using restriction endonucleases, recognition sites must be either side of the VNTR’s.
4. Must leave VNTR’s intact. VNTR’s are repetitive sequences of non-coding DNA.
   Can vary by:
   - Number of bases in the repeating sequence varies.
   - Number times the sequence is repeated.
   - Restriction endonucleases cut at recognition sites on either side of the VNTR’s.
   - Distance between recognition sites vary and are unique to each person.
   - Produces different sized fragments.
5. DNA fragments separated using (gel) electrphoresis.
   - Separates DNA fragments based on size / mass / charge.
   - Rate at which fragments of DNA move proportional to size / mass / charge.
   - Smaller fragments will travel further.
6. Detail of process for example: mixture put into wells on gel and electric current passed through.
   - DNA is hydrolysed by restriction endonucleases.
   - Hydrolysed DNA placed in well at one end of an agarose gel.
   - Gel is placed in a buffer.
   - Electrical current is passed through the gel.
   - DNA is negatively charged ( due to its phosphate group).
   - DNA fragments migrate toward the positively charged end of the gel.
   - Smaller DNA fragments migrate through the gel more quickly / further than larger fragments.
7. Immerse gel in alklaine solution / two strands of DNA separated / make DNA single-stranded.
8. Southern blotting / cover with nylon / absorbent paper (to absorb DNA)
9. DNA is fixed to nylon / membrane using UV light.
10. (Radioactively / fluorescently labelled) probe added which is complementary to VNTR.
    - Probes are 8-12 base pairs long.
    - Bases are complementary to DNA sequence / allele / gene / VNTR.
    - DNA and probe = hybridisation.
    - Probes usually either radioactively or fluorescently labelled.
11. (Areas with probe) identified using X-ray film / autoradiography (if radioactive probe). If fluorescent probe- UV light used to identify area with probe.

Question 19

What are the uses of genetic fingerprinting? (5 points).

Answer 19

1. Forensic science.
2. Determine genetic relationships.
3. Medical diagnosis of genetic disorders and cancer.
4. Determining fenetic variability in a population.
5. Animal and plant breeding; prevents inbreeding.

Question 20

Definition of DNA hybridisation.

Answer 20

Annealing / joining / binding of two strands of DNA / RNA from two different sources. Technique is used to identify complementary to an allele / gene.

Question 21

Definition of DNA probe.

Answer 21

Short- single-stranded sequence of DNA with a specific base sequence which has been fluorescently labelled / produced light and is complementary to an allele / gene.

Question 22

Definition of proliferation.

Answer 22

Means more cell division.

Question 23

Definition of assay.

Answer 23

Means to test the activity of a biological molecule, for example: testing the transcription rate / activity for a protein.

Question 24

How are radioactive probes used to identify alleles or genes?

Answer 24

Made up of nucleotides containing 32P.

Question 25

How are fluorescently labelled probes used to identify alleles or genes?

Answer 25

They emit light.

Question 26

Describe the method for genetic screening (9 points).

Answer 26

1. The DNA being analysed is amplified using PCR to make many copies.
2. The DNA is cut into fragments using a restriction enzyme.
3. The DNA fragments are separated out by size using gel electrphoresis.
4. An alkali is added to break the hydrogen bonds between complementary bases. This creates single strands.
5. A nylon membrane is placed on top of the gel. The DNA transfers onto the nylon. This is Souther blotting. The DNA is fixed on using UV light.
6. Radioactively / fluorescently labelled DNA probes with a complementary base sequence to the allele / gene of interest are added and bind to the allele is present. This is DNA hybridisation.
7. Excess probes are washed away.
8. If a radioactive probe is applied, radioactively exposes X-ray film, producing an autoradiograph so the DNA can be visualised.
9. If a fluorescent probe is applied, exposure to UV light allows the DNA to be visualised.

Question 27

Definition of gene therapy.

Answer 27

The treatment of genetic diseases by giving patients healthy copies of defective genes.

Question 28

Compare somatic gene therapy gaianst germline gene therapy (4 points).

Answer 28

1. Somatic: functional gene inserted into body cell. Germline: functional gene inserted into egg, sperm, zygote.
2. Somatic: vectors required, for example: retrovirus, adenovirus, liposomes. Germline: currently illegal so not tested.
3. Somatic: if successful only affected cells are recombinant. Germline: if successful, all cells are recombinant.
4. Somatic: functional gene is not passed onto children. Germline: functional gene will be passed on to offspring.

Question 29

Describe the method for somatic cell therapy (4 points).

Answer 29

1. Body cells are removed.
2. Functional alleles of gene are inserted into cell using a vector.
3. Cell is then re-inserted back into the patient’s body.
4. If a large number of body cells cannot be removed safely, genes are inserted directly into the affected tissues.

Question 30

Describe the method for germline gene therapy (2 points).

Answer 30

1. A corrected / functional gene is inserted into an egg cell that has been fertilised in vitro.
2. Embryo then develops and is then re-implanted into the mother’s womb.

Question 31

List the issues of somatic gene therapy (10 points).

Answer 31

1. Genetically modified viruses have been tried to get the allele into the genome, but the host becomes immune, so cells will not be accepted after the intial go.
2. Functioning allele of the gene is introduced into target cells.
3. Genetic manipulations are restricted to the actual parent.
4. Liposomes are used to get the allele into the genome, but they may be inefficient.
5. Any treatment is short-lived.
6. Treatment must be repeated regularly.
7. Treatment using vector could cause an immune response / rejection.
8. Harmful side effects from using vector, such as viruses.
9. It is difficult to gte the allele into the gemone in a functioning state.
10. Techniques to gte the gene to the target are needed or specified cells must be removed, treated and then replaced.