DNA Sequencing

Question 1

The Human Genome

Answer 1

• Every cell in the body (except gametes) are diploid in number (46 chromosomes)
• Each chromosome is a long chain of DNA that is divided into genes
• Each gene is a sequence of bases that code for a particular protein
• In a diploid cell, half the chromosomes have been inherited by the mother and the other half from the father
• This means that between the two chromosomes in a homologous pair, there will be two copies of each gene
• The exception being the sex chromosome in males
• Between the 23 pairs of chromosomes there are thought to be up to 30 000 genes
• This complete set is known as our genome

Question 2

Human Genome Project

Answer 2

• Through the human genome project, we know the base sequence for the entire genome
• Work is now going onto identifying the locations of genes on each chromosome and its exact role
• This will allow us to:
  1. Detect faulty genes
  2. Individualise treatment
  3. Replace faulty genes
  4. Transfer genes into bacteria

Question 3

DNA Sequencing - Synthetic DNA nucleotides

Answer 3

• This is the determination of the sequence of bases on the strand of DNA
• The base sequence of a particular gene can be determined by a method devised by Fred Sanger
• Nucleotides bond to a hydroxyl group (OH) of the previous nucleotide
• Sanger’s method involves using synthetic DNA nucleotides (dideoxynucleotides) which have been altered to no longer have a hydroxyl group attached
• So when these synthetic nucleotides bond to the new strand of DNA forming, it will prevent further elongation of the sequence from that point as there is no hydroxyl group for the next nucleotide to bond to

Question 4

DNA Sequencing - mixture

Answer 4

• The single stranded DNA sample to be sequenced (multiple copies) is placed into a mixture with lots of normal nucleotides and DNA polymerase enzyme
• Radioactive DNA primer added to the mixture. A strand of DNA with known bases that attach to the start of the DNA strands to start the process. (Required for DNA synthesis to begin as the DNA polymerase enzyme can only add new nucleotides to an existing DNA strand of which the primer provides)
• Added to this mixture will be one of the synthetic radioactive DNA nucleotides

(needs to be radioactive so that it would show up on the x-ray autoradiogram)

• There will be 4 flasks of reaction mixture with the addition of one of the synthetic nucleotides in each flask (A,T,C,G)
• Alternatively the synthetic nucleotides may have a different label attached (e.g. fluorescent colours) in which case they can all go in the same reaction flask
• The DNA polymerase will start replicating the supplied DNA sample
• But each time the synthetic nucleotide attaches to the growing DNA strand, it will stop the elongation process
• Eventually the entire strand of DNA will be broken into different sized fragments (like cutting a string into different lengths)

Question 5

DNA Sequencing - Agarose Gel

Answer 5

• The final 4 mixtures are then placed onto gel and the different lengths of DNA move through the gel by electrophoresis alongside each other
• The fragments will separate based on their size
• Knowing what the final base must be for each fragment, we can then determine by comparing the samples what the base sequence for that section of DNA must be

Question 6

DNA Sequencing - Uses

Answer 6

• By comparing DNA sequences, changes in the alleles can be detected
• This will show whether the person has the disease
• Or if a mutation (point mutations, insertions, deletions) has occured

Question 7

Electrophoresis

Question 8

After Electrophoresis

Answer 8

• After electrophoresis, the gel is exposed to X-ray film and an autoradiogram is made
• The sequence is read from the bottom of the gel to the top and is complementary to the sequence found on the original template strand