Manipulating Genomes

Question 1

PCR

Answer 1

1. DENATURATION
   95°C
   - DNA fragments mixed with DNA nucleotides and primers
   - this temp breaks the H bonds between complementary base pairs
   - The DNA strands separate
2. ANNEALING
   55°C
   - primers bind to complementary bases
   - required for replication of the DNA strands
3. ELONGATION
   72°C
   - DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double stranded DNA identical to the original sequence

Question 2

Gel Electrophoresis

Answer 2

1. DNA is amplified using PCR
2. DNA is cut into smaller fragments using restriction enzymes
3. DNA fragments are placed into the wells at the end of the agarose plate near the cathode (negative)
4. The plate is immersed in a buffer solution which helps maintain the pH
5. An electrical current is passed through the plate and DNA starts to separate as the negative phosphate group of DNA is attracted to the positive anode
6. The shorter DNA fragments move further than the longer fragments
7. Ethidium bromide is used as a stain to see the fragments

Question 3

Purpose of DNA profiling

Answer 3

DNA is used to identify individuals and species

• paternity tests
• identify new species
• forensics

Question 4

Southern blotting

Answer 4

• alkaline solution is added to the gel after electrophoresis and a nylon filter / nitrocellulose paper is placed over it
• DNA fragments ‘blot onto the filter’
• the alkaline solution denatures the DNA fragments so the strands separate and the base sequences are exposed
• a single stranded gene probe that has a radioactive label can then be applied to the filter and it will bind to the DNA in a highly specific pattern

Question 5

DNA profiling steps

Answer 5

1. DNA is prepared
2. DNA cut with restriction enzymes and loaded into the gel
3. Gel electrophoresis
4. Southern blotting

Question 6

Genetic engineering steps (basic)

Answer 6

1. Isolation of desired DNA fragment (using restriction enzymes or reverse transcriptase)
2. Multiplication of DNA fragment (PCR)
3. Transfer into organism using vector
4. Identification of cells with new DNA fragment
5. Growth/cloning

Question 7

Restriction endonucleases

Answer 7

Used to cut genes at specific base sequences (recognition sites)

Question 8

DNA ligase

Answer 8

Used to join together the cut ends of DNA by forming phosphodiester bonds

Question 9

Reverse transcriptase

Answer 9

Used to build double stranded DNA from single stranded RNA

Question 10

1. Isolation

Answer 10

Use reverse transcriptase
- mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
- double stranded DNA is formed in the template of the cDNA using DNA polymerase
- copy of DNA coding for original protein e.g. insulin

Use restriction endonucleases
- these cut up DNA
- some of these leave fragments with blunt ends, others with sticky ends

PCR primers are used to amplify the gene

Question 11

2. Insertion

Answer 11

• Insert the DNA fragment into a vector e.g. bacterial plasmid or weakened virus
  -restriction enzyme cuts DNA at specific recognition site
• annealing: DNA ligase joins DNA backbone back together

Question 12

3. Transformation

Answer 12

The transfer of DNA into suitable host cells can be done by:

• Heat shock treatment- bacteria alternated between 0°C and 42°C with calcium chloride, membranes become more porous
• Electroporation-high voltage pulse applied to cell to disrupt membrane
• Electrofusion- electrical fields help introduce DNA into cells
• Transfection- DNA packaged into a bacteriophage, which can then transfect the whole cell
• Recombinant plasmids- T1 plasmids are inserted into the bacteria which infects some plants

Question 13

4. Identification

Answer 13

• identify the host cells that have successfully taken up the gene by use of gene markers
• the colonies are shown under UV light and any cells that don’t glow have successfully taken up the plasmid because the fluorescent gene has been disrupted
  OR
  if antibiotic resistance gene was used then any cells that die have successfully taken up the gene

Question 14

5. Growth/cloning

Answer 14

Grow the cells from the colony that have the correct gene inserted in them

Question 15

Benefits of genetic engineering

Answer 15

• use for research e.g. diabetes, cancer
• get more vitamins and minerals into food such as rice / improve food health
• plants better adapted for environment
• vaccines
• improve crop yield- increase growth, pest resistance
  fewer pesticides used- better for environment and water

Question 16

Risks of genetic engineering

Answer 16

• no long term research of risks to humans
• prevents natural selection
• reduction in biodiversity- loss of native/original species
• risks of disease wiping out species
• contaminate ‘organic crops’
• antibiotic resistance / herbicides & pesticides
• exploitation of farmers
• companies use it to make profit (patents)

Question 17

Explain why a mutated allele may cause a genetic disease

Answer 17

• a mutated allele is a change in the DNA base sequence
• mRNA that is transcribed is wrong / different
• The amino acid is changed
• The protein produced is different (primary structure)
• This means that the protein can no longer function

Question 18

Somatic cell gene therapy

Answer 18

• harder to deliver genes. Must be ex vivo or in vectors (can be ineffective)
• can’t pass on new genes to offspring
• specialised cells are treated and don’t divide
• can’t pass on genes to other cells. Need to repeat gene therapy regularly (as specialised cells are replaced)
• allowed in humans

Question 19

Germline gene therapy

Answer 19

• delivering genes is easier as it is straight into germ cell
• can pass on new genes to offspring
• no need to repeat therapy as every cell and hence every new cell will contain a copy of new genes
• not allowed in humans for ethical reasons- danger of designer babies/eugenics

Question 20

Vectors used in gene therapy

Answer 20

• viruses
• liposomes
• plasmids

Question 21

Uses of DNA sequencing

Answer 21

1. Disease analysis- analyse risk of inheriting certain diseases (identify certain genes that increase risk)
2. Classification- evolutionary relationships derived from similarities in base sequences
3. Genetic engineering

Question 22

Bioinformatics

Answer 22

Creation of data bases that store information e.g. DNA sequences

Question 23

Computational biology

Answer 23

Use of bioinformatics in certain applications

Question 24

Sanger sequencing

Answer 24

• used gel electrophoresis
• radioactive labelled nucleotides
• not automated
• slower, inefficient
• labour intensive
• analyse gels by eye
• 4 reactions

Question 25

Automated Sanger sequencing

Answer 25

• used capillary gel electrophoresis • Florescent • Automated • Faster, efficient • Computer (not labour intensive) • Lasers to read DNA • Mixed together

Question 26

Gene sequencing has allowed us to:

Answer 26

- compare genomes between individuals and between species - identity/compare sequences of amino acids in polypeptides - develop synthetic biology

Question 27

Alternative methods to Sanger sequencing

Answer 27

- pyrosequencing - high throughput - massive parallel - next generation - nanopore