Manipulating Genomes

Question 1

Describe Fred Sanger’s DNA sequencing approach

Answer 1

• 4 dishes each with a single strand of DNA, DNA polymerase and a solution of the 4 bases
• Single strands of DNA broken up into chunks of every length
• A modified version of one of the DNA bases is added to each dish and tagged with a radioactive label
• Radioactive tag at the end of each length of DNA is read and pieces lined up in order of length
• Thousands of DNA fragments generated and passed through a gel by electrophoresis so fragments became sorted by length

Question 2

Describe the steps of pyrosequencing

Answer 2

1. A long length of DNA is cut into fragments using a nebuliser
2. These lengths are degraded into single-stranded DNA, they are the template DNAs and are immobilised
3. Sequencing primer is added and DNA is incubated with enzymes
4. One activated nucleotide (ATP, TTP, CTP or GTP) is incorporated into a complementary strand of DNA using the strand to be sequenced as a template
5. Tow extra phosphoryls released as pyrophosphate
6. The enzyme ATP sulfuryase converts pyrophosphate to ATP
7. The enzyme luciferase converts luciferin to oxyluciferin which generates visible light which can be detected
8. AMount of light is directly proportional to the amount of ATP available

Question 3

The procedure of DNA profiling

Answer 3

1. DNA obtained from individual
2. DNA digested with restriction enzymes which cut the DNA at specific recognition sites and cut it into fragments
3. Fragments separated by gel electrophoresis and stained
4. A banding pattern can be seen
5. DNA to which the sample is being compared with goes through the same process
6. Banding patterns between the two samples can be compared

Question 4

What type of DNA is analysed in DNA profilin?

Answer 4

Short-tandem repeats

Question 5

3 uses of DNA profiling

Answer 5

Forensic sciences - identify bodies etc.
Maternity and paternity tests - half of STR comes from mother, half from father
Analysis of disease - detects repeat sequences e.g Huntingtons disease and type of protein present e.g sickle cell anemia

Question 6

PCR relies on the facts that…

Answer 6

• DNA is made up of two antiparallel backbone strands
• each strand of DNA has a 5’ and a 3’ end
• DNA grows only from the 3’ end
• base pairs pair up in complementary pairs

Question 7

Steps of PCR

Answer 7

1. Sample of DNA mixed with DNA nucleotides, primers, magnesium ions and Taq DNA polymerase
2. Mixture heated to 94 degrees to break hydrogen bonds between base pairs, DNA becomes two single strands
3. Mixture cooled to 68 degrees so primers can anneal by hydrogen bonds to one end of each single strand. This gives a small section of double stranded DNA
4. Taq DNA polymerase bind to the end where there is double stranded DNA.
5. Temperature raised to 72 degrees to keep DNA as single strands
6. Taq DNA polymerase catalyses the addition of DNA nucleotides to single stranded DNA starting at the end with the primer in the 5’ to 3’ direction
7. When Taq DNA polymerase reaches the end, a new double stranded DNA has been generated
8. Repeat for many cycles

Question 8

Applications of PCR

Answer 8

Tissue typing - reduces risk of rejection for transplants
Detection of oncogenes - if type of mutation in a patients cancer is found, better treatment can be found
Detecting mutations - DNA analysed for mutations that could lead to a genetic disease
Identifying viral infections - PCR can detect viral genome among host cells
Monitoring the spread of infectious diseases
Forensic science - small quantities can be amplified for more tests
Research - more DNA, more reseach

Question 9

Why is electrophoresis used?

Answer 9

To separate DNA fragments

Question 10

What is the setup of gel electrophoresis?

Answer 10

An agarose gel plate is covered by a buffer solution. Electrodes are placed at each end of the tank so that an electric current can pass through. There is a positive end and a negative end.

Question 11

Steps of gel electrophoresis

Answer 11

1. DNA samples are digested with restriction enzymes to cut them into fragments. This is carried out at 35 degrees
2. A loading dye is added to the digested DNA
3. DNA is added to wells in the electrophoresis gel
4. Electrodes put in place and power turned on
5. DNA fragments move through gel at different speeds, smaller fragments travel faster

Question 12

How is electrophoresis used to separate proteins?

Answer 12

A charged detergent is used instead which equalises the surface charge on the molecules and allows proteins to separate according to their molecular mass.

Question 13

Examples of how DNA probes are useful in locating specific DNA sequences

Answer 13

• to locate a specific gene for use in genetic engineering
• to identify the same gene across different genomes when conducting genome comparison studies
• to identify the presence of a specific allele for a particular genetic disease

Question 14

Which enzyme catalyses the joining of sugar and phosphate groups within DNA?

Answer 14

DNA ligase

Question 15

What are the main stages of genetic engineering?

Answer 15

1. Required gene is obtained
2. A copy of the gene is placed inside a vector
3. Vector carries the gene into the recipient cell
4. Recipient expresses the gene

Question 16

There are 4 ways of obtaining the required gene for GE

Answer 16

1. mRNA obtained from cell where gene is expressed. Reverse transcriptase catalyses formation of complementary DNA using mRNA as a template. Dddition of primers and DNA polymerase can make it double stranded.
2. If nucleotide sequence of the gene is known, gene can be synthesised using an automated polynucleotide synthesiser
3. If sequence of gene is known, scientists candesign PCR primers to amplify the gene
4. DNA probe used to locate gene within genome and it can be cut using restriction enzymes

Question 17

How to place a gene into a vector for GE

Answer 17

• plasmids can be obtained e.g from obtained and mixed with restriction enzymes that will cut the plasmid at specific recognition sites
• Plasmid now has unpaired nucleotide bases called sticky ends
• if nucleotide bases, complementary to the sticky ends, are added to the gene to be inserted, the gene and plasmid should anneal catalysed by a ligase enzyme

A gene could also be sealed into a weakened virus that could carry it into the host cell

Question 18

4 ways to get the vector into the recipient cell for GE

Answer 18

1. Heat shock treatment - bacteria is subjected to periods of hot and cold in the presence of calcium chloride and walls become more porous
2. Electroporation - a pulse of electricity makes the recipient cell membrane more porous
3. Electrofusion - electrical fields help introduce DNA into cells
4. Transfection - DNA packaged into bacteriophage which can trannsfect the host cell

Question 19

What can be used to directly inserted gene into plant

Answer 19

A gene gun can be used. Small pieces of gold or tungsten coated with the DNA are shot into the plant cells

Question 20

What is somatic cell gene therapy?

Answer 20

Gene therapy by inserting functional alleles into body cells. It only affects certain cell types and the alterations are not passed to the patient’s offspring.

Question 21

What are liposomes?

Answer 21

Small vesicles that can be used in gene therapy to introduce a functioning allele into a cell.

Question 22

Potential problems with using viruses as gene delivery agents

Answer 22

• may provoke an immune or inflammatory response
• Can only be used once as the patient may become immune to the virus
• Virus may insert allele in a location that disrupts a gene in regulating cell division which could lead to cancer
• virus may insert gene in a location that disrupts the expression of other genes

Question 23

What is germ line gene therapy?

Answer 23

Gene therapy by inserting functional alleles into gametes or zygotes. Offspring will also inherit the foreign allele.