Manipulating Genomes

Question 1

What are the 5 main stages of producing a DNA profile/fingerprint?

Answer 1

1. Extraction - DNA is extracted using a tissue sample and amplified using PCR
2. Digestion - restriction enzymes cut DNA into fragments at specific points
3. Separation - electrophoresis separates cut fragments of DNA to form a pattern
4. Hybridisation - radioactive or fluorescent DNA probes bind with VNTR’s
5. Development - DNA fragments are visualised as a pattern of bars

Question 2

What are the basic principles of DNA sequencing?

Answer 2

1. DNA is mixed with a primer, DNA polymerase, nucleotides and terminator bases
2. Double stranded DNA is split into single strands and primers anneal to the DNA
3. DNA polymerase adds nucleotides to the single stranded template to build new DNA strands
4. When a terminator base is randomly added instead of a base, DNA synthesis stops
5. This produces all the possible fragment lengths of DNA
6. DNA fragments are separated according to length, eg by gel electrophoresis
7. Fluorescent markers identify the final base of each fragment and lasers detect the sequence order
8. The sequence order on the new, complementary DNA strand is used to determine the order on the original DNA strand

Question 3

What are the key stages of gene transfer in genetic engineering?

Answer 3

1. The desired gene is identified and isolated
2. Multiple copies of the gene are made using PCR
3. The gene is inserted into a vector
4. Vector delivers the gene into cells
5. Cells with the new gene are identified, such as by using marker genes
6. Cells with the new gene are cloned

Question 4

What are the steps in PCR?

Answer 4

1. Denaturation - heat the DNA to 95 degrees to break the hydrogen bonds between complementary strands and separate them
2. Annealing - cooling to 55 degrees so primers can bind to DNA strands by forming H bonds
3. Extension - heating to 72 degrees for DNA polymerase to synthesise new DNA

Question 5

How can you tell whether bacteria have been successfully genetically modified using marker genes?

Answer 5

1. A marker gene that causes them to exhibit a specific trait, like antibiotic resistance, may be incorporated into the vector
2. A marker gene that codes for a fluorescent protein that can be seen under UV light, like GFP, may be incorporated into the vector
3. A marker gene may be inserted within the GFP gene, preventing fluorescence if it is successfully incorporated