Molecular Diagnosis: Lect 7-9 Flashcards
Linkage analysis
- to identify a genetic loci that is assoc. w/ a disease.
- gene of interest must be linked to a known marker locus (polymorphic marker) performed by recombination mapping.
- determines carrier status in AR
- Challenges: Recombination event and Locus heterogeneity.
Unlinked-
Linked-
gene/markers far apart on the same chrom; show independent assortment. show recomb freq of 50%
genes/markers on the same chrom and are close to each other; show recomb freq of
Direct detection
-detect what is asked for:
Southern, PCR-RFLP, ASO-PCR, PCR sizing, ASO blot, FISH, Northern, Western.
-able to query entire genome:
G-banding, Array CGH, SNP chip, expression arrays (cDNA), spectral Karyotype(SKY FISH), Next Gen sequencing.
Allele Specific Oligonucleotide probes: ASO probes
~15bp; bind to a single allele of a gene
- less useful if disorder exhibits allelic or locus heterogeneity.
- diagnosis of CF and Hemochromatosis.
ASO PCR
Allele specific PCR; used to detect single nucleotide changes.
+/+ homo nl
+/- hetero
-/- homo rec`
PCR:
Multiplex PCR:
used to detect 3X expansion; Huntington’s (>40 repeats), Fragile X, Myotonic dystrophy.
- no PCR product formed if exon is missing
- Multiplex PCR- dx DMD in affected boys; detects deletions. Won’t detect carrier mothers/CNV . Presence of all genes exons.
G-banding/Karyotype Analysis
- Giemsa stain to A-T rich region (dark bands)
- can detect changes in chrom number (aneuploidy).
- can detect very large deletions, insertions, rearrangements. >5Mb - 10Mb
- unable to resolve smaller chrom changes/low resolution
FISH:
two types?
- better resolution than G-banding
- cells don’t need to be in metaphase
- Gene/Chrom specific probe:
- hybridizes to a specific locus on a chrom; identifying submicroscopic deletions, translocations, duplications. - SKY-Spectral karyotyping :
- paints each chrom a diff color; characterizes complex chrom rearrangements/translolcations and origin of rearranged genetic material (cancer).
- small deletions are invisible
Velocardiofacial syndrome-VCFS
DiGeorge; 22q11.2 deletion syndrome.
- abnl pharygeal arch development, defective parathyroid dvlp, thymus, conotruncal heart, Tetralogy of Fallot.
- seen by G-banding but not always clear. Easier to detect w/ FISH and allows for better resolution but must know what to look for.
Array comparative genomic hybridization
- allows detection of copy number changes through the genome in a single hybridization.
- detect amplifications and deletions = CNV
- does not detect balanced rearrangement.like the Philadelphia chrom 9 and 22(SKY-FISH can detect this)
- doesn’t detect amplification above a certain number.
ex: tumor cell vs. nl cell; tumor genetics; trisomy 18.
- –compared to reference standard
SNP chips
- single bp changes, 1000s of polymorphism and point mutations in a single experiment.
ex: uniparental disomy, drug metabolism CYP450 array
cDNA microarray:
- “expression arrays”; representing a set of known transcribed seq.
- identifies a set of genes expressed in a cell or tissue
- compares gene expression btwn cell or tissue.
- cDNA made by Rev transcriptase from mRNA
-if a gene is overexpressed by cDNA microarray, the Northeblot is used to confirm it.
Sanger (dideoxy) sequencing:
- a method to obtain the bp seq of a piece of DNA
- random incorporation of dideoxynucleotide terminates the synthesis of the DNA strand.
Next Gen sequencing
-hybridizes single stranded DNA segments to primer sequences on a microarray chip, and uses massively parallel genomic sequencing to sequence all of the segments at the same time.
Western Blots
-detect and quantify the amount of a particular protein/labeled Ab system
