Mutation Detection

Question 1

Base substitution can be:

Answer 1

Transition and Transversion

Question 2

Pyrimidine to Pyrimidine
Purine to Purine

Answer 2

Base substitution ‘Transition’

Question 3

Pyrimidine to Purine

Answer 3

Base substitution ‘Transversion’

Question 4

Types of mutation?

Answer 4

Silent/synonymous
Missense (conservative & nonconservative)
Nonsense
Framshift

Question 5

Detection for known mutations:

Answer 5

• Allele specific oligonucleotide hybridization
• Multiplex Ligation Dependent Probe Amplification
• DNA Sequence
• DNA Microarray

Question 6

• Known mutation
• Also known as the “Dot-Blot” method
• Specific binding of the chromogenic probe to target DNA (with color = mutation)

Answer 6

Allele specific oligonucleotide hybridization

Question 7

• Known Mutation
• 50 different probes for 50 different genes
• Detects Deletion and Duplication
• unequal peaks = mutation, not amplified

Answer 7

Multiplex Ligation Dependent Probe Amplification

Question 8

• Known Mutation
• Base on the DNA sequence (compared to normal DNA)
• Different sequence = mutation
• Maxam Gilbert (bottom to top)
• Dye terminator

Answer 8

DNA sequencing

Question 9

• Known Mutation
• Probes are present in array, samples added to each wells
• Many colors, different samples, different fluorescent

Answer 9

DNA Microarray

Question 10

• Unknown mutation
• Detects 80-90%
• Fragment size limit 150-200 bp
• Different folding patterns = Different migration pattern = mutation
• secondary structure of sSDNA

Answer 10

Single Strand Conformation Polymorphism

Question 11

• Unknown Mutation
• modification of AGE
• Uses denaturing chemicals ( Formamide and Urea)
• Top = lowest denaturing condition
• Bottom = highest denaturing condition

Answer 11

Denaturing Gel Gradient Electrophoresis

Question 12

• Unknown Mutation
• Modification of DGGE
• Use temperature instead of denaturing chemical
• More reproducible and reliable

Answer 12

Temperature Gradient Gel Electrophoresis

Question 13

• Unknown Mutation
• Uses diff restriction cut sites to detect mutation
• Same principle with DNA Fingerprinting
• Change cut sites (different fragments) = mutation
• Detected mutations affect palindromic cut sites

Answer 13

Restriction Fragment Length Polymorphism

Question 14

• Unknown Mutation
• Homoduplex and Heteroduplex (bulge)
• No Heteroduplex= no mutation (no bulge)
• Base substitution not easily detected
• Homoduplex stays at the lower portion = mutant

Answer 14

Heteroduplex analysis

Question 15

• If wild type and homoduplex have the same migration
• Heteroduplex bulge cutted (bands)
• Lane with bands = mutation

Answer 15

Single stranded Specific Nuclease

Question 16

Detection for unknown mutation:

Answer 16

Single strand conformation polymorphism
Denaturing gel gradient Electrophoresis
Temperature gradient gel Electrophoresis
Restriction Fragment Length Polymorphism
Heteroduplex analysis