Molecular Diagnosis

Question 1

Recombination frequency

Answer 1

• If two loci are close together on a chromosome, the chances of recombination are LOW
• Loci that have low recombination frequency →LINKED LOCI

Question 2

Restriction Fragment Length Polymorphism

Answer 2

RFLP

palindrome restriction sites

If the mutation interferes with a restriction site, then RFLP allows Direct detection

Ex) Sickle cell anemia are affected by the mutation at the actual site for the restriction enzyme –allows direct test

Question 3

ASO

Answer 3

Allele Specific Oligonucleotide probes
Can be used with PCR

Short sequences (~15bp) specifically bind to a single allele of a gene

***Possible only if the exact mutation in the gene has been identified

Ex) used for diagnosis of cystic fibrosis and hemochromatosis

• w/ PCR to detect triplet expansion repeats

Question 4

MPLA

Answer 4

Multiplex Ligation-Dependent Probe Amplification

-Determines Heterogeneity

Detect insertions, deletions and single base pair (ASO) changes

Spike (hills and valley analysis)

1. Only one primer pair is used
2. Only ligated probes will be amplified
3. Less cycles of PCR amplification are allowed

Question 5

G-Banding

Answer 5

Karyotype analysis

binding of Giemsastain to A-T rich regions of the genome. (dark bands are A-T regions, and light bands are G-C rich regions).

***Insertions, deletions, and rearrangements on the several or manymega-base (Mb) level

Question 6

FISH

Answer 6

Fluorescent in situ hybridization

Fluorescently labeled probes can be used to hybridize partially denatured chromosome spreads

–Hybridizes to a specific locus (only one locus)

**For identifying submicroscopic deletions, translocations and duplications of genes at about the ~100,000 bp level

Ex) 22q11 detection

Question 7

SKY FISH

Answer 7

Spectral karyotyping or chromosome painting

**Useful for characterizing complex chromosomal rearrangements and identifying the chromosomal origin of rearranged genetic material

Question 8

Array CGH

Answer 8

Array Comparative Genomic Hybridization

Technique that allows detection of DNA sequence copy number changes throughout the genome in a single hybridization

**CGH may detect CNV (copy number variants) compared to “normal”
–Deletions (very small)
–Duplications (very small)

Ex) gain for X chromosome and loss or Y

Question 9

Next Generation Sequencing

Answer 9

Massively parallel genomic sequencing on a chip

Single stranded DNA segments to be sequenced are hybridized to primer sequences that are attached to a microarray chip.

Question 10

SNP Chip

Answer 10

Genomic DNA fragments hybridize to DNA spots adhered onto a microarray slide where each spot represents various alleles of a gene

Ex) The Roche AmpliChip CYP450 Array is a device that can be used to determine genotypes for alleles of selected CYP genes.

Question 11

cDNA microarray

Answer 11

Expression Arrays representing a set of known transcribed (expressed) sequences.

1.May be used to identify a set of genes or all genes that are expressed in a cell or tissue

2.May be used to compare gene expression between cell or tissue samples
–Comparative

Ex) characterize differences between the genes expressed in cancer tissues compared to non-cancerous tissues