Molecular Diagnosics

Question 1

RFLP (restriction fragment length polymorphism)

Answer 1

Detects: mutation in restriction site
Advantage: compare DNA of 2 unrelated individuals
Disadvantage: mutation must create or destroy a restriction site
Dx: Sickle Cell

Question 2

ASO (Dot Blot) Assay

Answer 2

Detects: single base pair changes, or just a few bases (exact KNOWN mutations only, like things that are common in a population)
Disadvantages: less useful for disorders w/ increased allelic/locus heterogeneity, doesn’t confirm carrier status
Dx: CF, hemochromatosis

Question 3

PCR Sizing (gel electrophoresis)

Answer 3

Detects: size of PCR product
Dx: triplet expansion repeat disorders (Fragile X, Huntinton’s, Myotonic Dystrophy)

Question 4

Karyotype Analysis

Answer 4

Detects: rearrangements, ploidy status, insertions and deletions 5Mb-10Mb
Disadvantages: cannot detect MICROdeletions, lower resolution to CGH; G = Giemsa stain which requires chromosomes to be in Metaphase

Question 5

FISH (chromo/gene-specific probes)

Answer 5

Detects: confirms suspected small deletions or translocations, sometimes duplications
Advantages: facilitate automation of genetic analysis, evaluate effects of chemical mutagens, microdeletions
Disadvantages: does not scan entire genome
Dx: DiGeorge (VCFS)

Question 6

SKY-FISH, M-FISH

Answer 6

Detects: origin of chromosome rearrangements
Advantages: facilitate automation of genetic analysis, evaluate effects of chemical mutagens, microdeletions

Question 7

Array CGH (Comparative Genome Hybridization)

Answer 7

Detects: copy # variants (CNV) of DNA so deletions/duplications
Advantages: map location of genomic duplications/deletions btwn pt DNA and reference DNA, detects changes > 1Mb of DNA
Dx: useful in tumor genetics

Question 8

Sanger Sequencing

Answer 8

Detects: obtain a base pair sequence of a piece of DNA

*aka dideoxy or Chain Termination

Question 9

SNP Chip (Array)

Answer 9

Detects: SNP (single bp changes, polymorphisms and point mutations)
Advantages: thousands of probes may be searched in a single experiment, can detect susceptibility to adverse drug rxns (CYPs)
*can detect uniparental disomy (if parents have had SNP chip

Question 10

cDNA Array (expression array)

Answer 10

Detects: set of genes or all genes that are expressed in a cell/tissue
Advantages: microarray w/ features representing a set of known expressed sequences; compare gene expression btwn cell/tissue samples

Question 11

Protein Arrays

Answer 11

Detects: proteomics, protein functional analysis, ab characterization, rx development
Advantages: drug development; large # protein targets tested, drugs can be synthesized en masse and rapidly tested

Question 12

Southern Blot

Answer 12

(DNA)

identify gene sequence: mutations and polymorphisms

Question 13

Northern Blot

Answer 13

(mRNA)

study gene expression: quantitation and alternative splicing

Question 14

Western Blot

Answer 14

(Proteins)

identify: antibodies to an infectious dz

Question 15

Next Generation Sequencing

Answer 15

Massively parallel genomic sequencing