Genetic studies of cancer Flashcards
Molecular diagnostic methods examples x4
PCR, MLPA, SNP analysis + SNP analysis by qpcr
MLPA stands for
Multiplex Ligation - mediated Probe amplification
Ligase chain Reaction principle
DNA product formed by ligating 2 probes together on target DNA = signal if template present
Ligation chain Reaction visualization
Gel or labelled Probes
Ligase chain Reaction method
1) DNA denatured
2) Probes bind to target
3) DNA ligase join new strand to template =dsDNA
4) Repeated heating+ cooling = amplification
5) Ligated DNA detected
LCR Pros
Highly Quantifiable, Limited non- specific products forming
MLPA Principle
Multiple LCR followed by PCR detection
MLPA probes
3’ = stuffer sequence = diff in each probe
5’ = same primer recognition site on both
Diff hybridization Sequences
40+ pairs diff lengths
MPLA method
1) Denaturation
2) hybridization of probes
3) Ligation of 2 probes by ligase
4) PCR: Primers ( 1 set) bind recognition site + amplification of ligated products
5) separate products by electrophoresis
MLPA electrophoresis
capillary electrophoresis
Forward probe fluorescently labelled
Uses of MLPA
mapping large chromosomal deletions + imbalance screening, Genome screen for unknown del.
MLPA results
compare peak height with control sample shows Sequences with aberrant copy number
SNP
single - nucleotide Polymorphism = Silent or pathogenic
Illumina Bead Chip does?
Total DNA amplified in short Sections
SNP method: BeadChip
1)Each bead has short capture sequence with stops 1 base before known SNP
2) Captures Region of DNA
3) DNA poI extends from capture sequence
4) each nucleotide labelled with diff colour t chain terminator= only 1 base added
