FHMP 010 Molecular biology and medicine

Question 1

what were the steps used to first sequence the human genome?

Answer 1

1. fragmentation, digest the DNA into smaller pieces
2. Separation, separation the DNA fragments
3. Cloning. clone the DNA
4. Sequence the DNA
5. Assemble the fragments

Question 2

describe the process of fragmentation

Answer 2

• use restriction enzymes (endonuclease) to break the DNA at specific sequences
• sticky ends are formed when they are both cut by the same restriction enzyme, so the ends are complementary
• ligase enzyme ligase / stick DNA bases

Question 3

describe the process of separation

Answer 3

• DNA fragments are separated using gel electrophoresis
• an electric current is applied across the gel so 1 end has a + charge and the other end a - charge
• DNA is negatively charged so it moves towards the positive end
• The DNA fragments are separated by size, smaller fragments move faster/further

Question 4

describe the process of cloning the DNA

Answer 4

• bacterial plasmids have 3 regions:
  = polylinker sites where restriction enzymes can cut open the plasmid and insert foreign DNA
  = drug resistance gene in plasmid to be inserted
  = replication origin which allows the plasmid to replicate
• Eco R1 is used to cleave the plasmid at the polylinker site, producing sticky ends (complementary to to foreign DNA fragment
• the foreign DNA fragments and the cleaved plasmids are mixed together and the sticky ends hybridise
• DNA ligase joins the hybridised DNA with the formation of new phosphodiester linkages = called recombinant DNA
• then bacteria treated with calcium chloride to make membranes permeable to the plasmids
• bacteria cells poured onto nutrient agar with ampicillin so only bacteria with the plasmid with resistant gene will survive
• the correct bacteria then multiply and clone the DNA in the plasmids

Question 5

describe sequencing of DNA

Answer 5

• order of bases on the DNA fragments are read

Question 6

describe the process of fragment assembly

Answer 6

computers are used to assemble the fragments

Question 7

how is cloning DNA/genes using bacteria used in medicine?

Answer 7

• insulin = produce from pig pancreas for diabetes
• growth hormone = take from pituitary for hypopituitarism
• Factors VIII and IX = take from blood for Haemophilia A and B

Question 8

what are some problem with using bacteria to clone genes?

Answer 8

insulin = allergy (non-human source), religious concern
growth hormones = scarce, ethical concerns
Factors VIII and IX = viral infections from blood

Question 9

what is cDNA?

Answer 9

A complementary strand of DNA is synthesised from an RNA template by the enzyme reverse transcriptase.

Question 10

What is southern blotting?

Answer 10

A technique that uses DNA probes/radioactive labels to detect the presence of specific DNA in restriction fragments separated by electrophoresis.

Question 11

What is sickle cell anemia?

Answer 11

A mutation in a gene causes a problem affecting the haemoglobin of the red blood cells.,
A genetic disorder causes red blood cells to be rod/sickle-shaped.
This causes them to get stuck in capillaries and the bodies cells become deprived of oxygen = hypoxia

Question 12

how can you diagnose sickle cell anaemia using southern blotting?

Answer 12

• restriction enzyme Mst 2 cuts DNA at specific sequence
• in normal HbA, there are 3 of these specific sequences in the beta chain
• in HbS there is a single base change so the base sequence doesn’t exist in the middle of the gene
• HbA with Mst 2 will produce 2 fragments of DNA and 2 strands
• HbS with Mst 2 will produce only 1 longer strand of DNA and 2 strands
  so normal people = 2 light fragments
  sufferers = 1 heavier fragment
  carriers = 1 light and 1 heavy fragment

Question 13

what is trimethylaminuria?

Answer 13

• fish odour syndrome
• a rare metabolic disorder that causes a defect in the normal production of the enzyme FMO3
• when FMO3 is not working properly or not enough is produced, the body looses the ability to properly convert trimethylamine (TMA) from precursor compounds in food digestion into trimethylamine oxide (TMAO) through N oxidation
• so TMA builds up and is released in sweat, urine and breath giving off a fish odour

Question 14

how can Trimethylaminuria diagnosed?

Answer 14

• the gene for FMO3 has several different exons separated by introns
• PCR is used to amplify each exon
• each exon is sequenced through ddNTP chain termination
• the code is read and checked for the mutation of CCC to CTC coding for leucine/ uses DNA probes