L11: Targeted Variant Detection

Point Mutations

• Gene mutations involving one or a very few base pairs.

• Not detectable by the cytogenetic method.

• May result in phenotypic change

Duplications

copy of one or more nucleotides are inserted directly 3' of the original copy of that sequence

Inversion

reverse complement of the original sequence

Mutation Detection: Hybridization-Based Methods

– Allele-specific Oligomer Hybridization (ASO)

– Melt curve analysis (MCA)

– Heteroduplex analysis (HA)

– Array technology

Allele-specific Oligomer Hybridization (ASO)

• Dot blot method, control/test specimens spotted on nitrocellulose and labeled oligonucleotide probe (~20 bp) is

hybridized to immobilized specimen.

• Under a specific assay condition (stringency) the ss probe will not bind if there is even 1 nucleotide mismatches.

Melt Curve Analysis

usually a postamplification step of qPCR.

dsDNA specific dye (EtBr SyBr Green) will fluoresce brightly when bound to DNA and by slowly increasing the temperature, dsDNA separates into single strands losing the dye, resulting in loss of fluorescence which is detected and graphed by the instrumentation

Melt curve Tm

Every sequence has a characteristic Tm and indicates which sequence is present.

A mismatch will lower the Tm of the duplex.

Melt Curve Analysis Specificity

increased using FRET probes specific for the sequence position being analyzed

Heteroduplex Analysis

formed from non-complementary ss hybridization to another

resolved from the homoduplex by electrophoresis

SSP-PCR

detects point mutations and SNPs.

3’ end nucleotide primer anneals to one sequence version and is critical for the

PCR product indicates presence of corresponding mutation or polymorphism in the template

Allelic Discrimination

fluorescent signal for mutant or normal sequence, performed on qPCR

Allelic Discrimination Probes

uses two TaqMan probes (normal and mutant), each with an oligonucleotide with a 5′ reporter dye and a 3′ quencher dye.

probe matching the test sequence is digested by the enzyme, releasing the reporter dye

MLPA

Amplification of a probe is dependent on a ligation, only occurs if target DNA is present in the sample.

• For a short sequence of target DNA, forward and reverse primer sequence are designed and hybridized against the target DNA and subsequently ligated.

MLPA Product

functional PCR strand appears if ligation occurs

Amplification only happens if target DNA is present

amount of PCR product (fluorescent signal) is proportional to the amount of target DNA present in the sample

RFLP

Fragments sizes vary as a result of the mutation changing the nt sequence for restriction enzyme recognition sites.

Number and sizes of the restriction fragments of test DNA cut with restriction enzymes are compared with the number and

sizes of fragments expected from a reference restriction map