EKQu's |DNA technology

What is a gene?

A section of DNA that codes for a single polypeptide

Define genome

All the DNA in an organism

Define proteome

All the proteins made by an organism

Describe how whole-genome shotgun sequencing is used to determine the DNA sequence of an organism.

DNA is cut into small, easily sequenced sections and computer algorithms are used to align the overlapping segments

Why is it easier to determine the proteome of prokaryotic organisms?

·Most prokaryotes have just one circular piece of “naked” DNA

· They do not have any introns

Why is it more difficult to determine the proteome of complex organisms?

They have non-coding genes that have a role in regulating other genes.

Name the enzyme used to cut open a plasmid.

Restriction endonuclease

Name the enzyme used to insert a gene into a plasmid.

DNA ligase

What is the role of DNA ligase in the production of plasmids?

Allows the annealing of phosphodiester bonds

How is DNA broken down into smaller fragments?

Restriction endonuclease cuts the DNA at a specific base sequence

Explain why the scientists used the same restriction endonuclease enzymes on each DNA sample.

So they cut at the same base sequence to get the fragment with the required gene.

Describe how a gene can be isolated from human DNA.

Use a restriction endonuclease to cut DNA at a specific base sequence

What is meant by a vector?

A carrier of foreign DNA into another organism.

Explain how sticky ends are useful in genetic engineering.

They join two pieces of DNA by complimentary base pairing.

Why do scientists use a marker gene, specifically GFP (green fluorescent protein)?

To identify organisms that have taken up the plasmid as not all will. The organisms that have taken up the gene will glow allowing them to be identified easily under UV light.

Explain why it is easier to obtain a gene from mRNA than from DNA removed from cells.

· mRNA will be present in large amounts of cells making the protein

· It does not contain introns · It only codes for a single protein.

Explain why a gene from an insect can be expressed in a plant.

· The genetic code is universal

· So the insect gene can be transcribe

· So the gene can be translated as the mechanism is the same in all organisms

Why is it better if plasmids are injected into eggs rather than the adult organism?

The gene gets into all/most cells of the organism

Explain why PCR would be carried out.

To increase the amount of DNA

Why is it an advantage the enzyme used in PCR is thermostable?

It would not denature when heated to 95

What are DNA primers?

Short lengths of single stranded DNA.

Why are two different primers required in PCR?

The DNA sequence is different at the start compared to the end.

Explain why primers only bind to specific DNA fragments.

They have specific base sequences that are complimentary to the fragments

What is the role of DNA polymerase in PCR?

Joins nucleotides to produce complimentary strands of DNA

Explain the function of primers in PCR.

They mark the region of DNA to be copied. The enzyme needs a starting strand to add nucleotides to

Explain why DNA heated to 95 in PCR.

To break the hydrogen bonds to separate the strands of DNA.

Explain why the mixture is cooled from 95 to 55 in PCR.

To allow hydrogen bonds to reform and to allow the annealing of primers.

Describe how to calculate the number of DNA fragments after PCR cycles.

2n where n = number of cycles.

Give reasons why DNA replication might stop in PCR.

· Limited number of nucleotides

· Limited number of primers

· DNA polymerase eventually denatures

What is a DNA probe?

A short single strand of DNA that complimentary base pairs with a gene

Explain why DNA on the nylon membrane is treated to form single strands.

So the DNA probe binds

Explain why radioactive DNA probes are used to locate specific DNA fragments.

· DNA is invisible on the gel so being radioactive allows detection

Explain how the use of a gene probe could detect the presence of a mutant allele.

· Probe will attach to the mutant allele

· It will attach to one DNA strand as a result of complimentary base pairing

· Radioactivity is detected on film if the mutant allele is present.

What is meant by “gene therapy”?

Introduction of a healthy gene

Give advantages of using a virus in gene therapy.

· It can enter cells

· It targets specific cells

· It replicates in cells

Suggest why bone marrow for a transplant is obtained from a genetically matched donor.

· To prevent an immune response (rejection

Give two features of the structure of proteins that enable them to be separated by gel electrophoresis.

· Mass

· Charge

Explain how gel electrophoresis separates fragments of DNA.

· DNA fragments move towards the anode because they are charged.

· They move at different rates due to their size/charge.

Explain how the control lane of a gel electrophoresis allows the size of fragments to be determined.

It has DNA fragments of known lengths to compare the fragments of unknown length to.

Explain why base pairs are a suitable way of measuring the length of a piece of DNA.

DNA is made of base pairs.

Each base pair is the same length.

Explain how genetic fingerprinting allows scientists to identify the father of a child.

All bands in cub which don’t come from mother must be in father’s DNA fingerprint.

Suggest how genetic fingerprints might be used to ensure only unrelated animals breed.

Select pairs with dissimilar DNA fingerprints

Describe how a gene could be isolated from an animal cell and introduced into a crop plant

1. Gene is identified using a gene probe

2. Cut using restriction endonuclease

3. At specific base pairs (recognition site)

4. Leaving sticky ends

5. Cut vector using same restriction endonuclease

6. Join using DNA ligase

7. Introduce vector to crop plant cells

Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected

1. Isolate wanted gene

2. Using a restriction endonuclease

3. To get DNA and sticky ends

4. Use DNA ligase to join wanted gene to plasmid

5. Include a marker gene (e.g. antibiotic resistance)

6. Add plasmid to bacteria to grow colonies then replica plate onto medium where the marker gene is expressed (e.g. containing antibiotic)

7. Colonies not killed have antibiotic resistance gene (probably) the wanted gene

Describe the polymerase chain reaction.

1. Heat DNA to 90

2. To separate the strands

3. Add primers

4. Add nucleotides so they attach by complimentary base pairing

5. Cool so that primers bind to DNA

6. DNA polymerase joins nucleotides

7. Cycle is repeated many time

Describe how genetic fingerprinting may be carried out

1. DNA extracted from sample

2. DNA cut using restriction endonucleases

3. DNA fragments separated using electrophoresis according to length / mass

4. Transfer to nylon membrane / Southern blotting

5. DNA made single-stranded

6. Apply probe that is radioactive/fluorescent

7. That is complimentary to VNTR/minisatellites

8. Areas with probe are identified using autoradiography/x-ray film.

9. The pattern is unique to every individual.