Using DNA sequencing to diagnose single gene disorders

Question 1

Types of Genetic variants:
Synonymous?

Nonsense?

Missense?

Insertion/Deletion: What happens when it’s in a multiple of 3 or not?

In what order do these variants become increasingly likely to cause a pathology?

Answer 1

• Mutation that doesn’t change the amino acid
• Mutation causes premature stop = Shorter protein
• Mutation changes the amino acid = Malfunctioning protein
• • Multiple of 3 amino acids = NO effect on the rest of the mRNA
  • NOT a multiple of 3 = FRAMESHIFT
• Synonymous → Missense → Nonsense/Insertion/Deletion

Question 2

How does PCR work?

How does Sanger work?

How does Next Generation Sequencing (NGS) work?

Answer 2

• Attach primers to the wanted section of DNA, heat DNA to break strands, add DNA polymerase and primers to form new double strands, then repeat over and over again to produce millions of copies
• Primer and DNA polymerase added, with Deoxyribose AND Dideoxyribose nucleotides
  o Dideoxyribose nucleotides can’t be extended further, so once attached, the sequence stops
  o Forms different length sequences with different colours, which is separated by electrophoresis
• DNA is fragmented and bound to adapter sequences, which is then bound
  to a captured framework

Question 3

Sequencing-bases Tests:
SINGLE GENE:
How many genes involved?

Cost?

Pros?

Cons?

GENE PANEL:
How many genes involved?

Cost?

Pros?

Cons?

WHOLE EXOME:
How many genes involved?

Cost?

Pros?

Cons?

WHOLE GENOME:
How many genes involved?

Cost?

Pros?

Cons?

Answer 3

• 1
• Very cheap
• Cost, Speed, Reliability
• Limited to 1 gene

• 1 to 100
• Cheap
• > 1 gene, Cost, Can detect mosaicism
• Limited to selected genes

• EXONS ONLY
• Moderate
• Quick, Can choose genes to test
• Limited

• EXONS + INTRONS
• Expensive
• Tests all genes
• Cost, Speed, Difficult to interpret (large data volume)