Molecular Diagnostics

explain the concept of linkage using RFLP and a pedigree; include the effect of a recombination event

1) obtain DNA samples and analyze by linkage to RFLP marker 2) read the gel & compare fragments of known diseased persons to potential 3) carrier status may be determined by linkage analysis for an autosomal recessive disorder (slide 7)

describe the mutation of cystic fibrosis & best molecular diagnostic

3 base pair deletion most commonly F508 ASO probe is useful for identification

what analysis identifies detection of subtle translocations

FISH

describe chromosome banding

G-banding is the most common type of karyotype analysis staining: dark bands are A-T, light are C-G Limitations: requires highly experienced cytologists to differentiate between chromosomes

MLPA

Multiplex Ligation Dependent Probe Amplification allows hundreds of sites to be tested and identified in a single reaction detects insertions, deletions, and single base pair (ASO) changes. uses a universal primer for amplification & each product is separated by size (see slide 40) two key points: 1) only ligated probes will be amplified 2) less cycles of PCR amplification are allowed

PCR methods: allele-specific PCR, Repeat expansion PCR

PCR allows analysis of DNA from any cellular source, creating primers for a target set of DNA that is then amplified to many more copies (exceeding 1 million) It had rapid turn around time Allele-Specific PCR is used to detect a single nucleotide change Repeat Expansion PCR detects triple repeats in autosomal dominant disorders, such as Huntington's & Myotonic Dystrophy, X-linked disorders, such as Fragile X syndrome

describe karyotyping

Process: peripheral blood sample phytohemagglutinin stimulated cell division cells arrested in metaphase by colchicine cells placed in hypotonic solution: swell & burst chromosomes fixed and stained staining: dark bands are A-T, light are C-G G-Banding (Giemsa staining) is the most common USE: aneuploidy, large translocations, insertions, deletions Limitations: does NOT detect microdeletions EXAMPLE: Trisomy 21

what analysis identifies characterization of complex rearrangements

SKY FISH

describe SKY

SKY or M-FISH: uses whole chromosome specific probes labeled with unique colored fluorescent dyes specific to each chromosome USES: characterizing complex chromosomal rearrangements & identifying the chromosomal origin of rearranged genetic material Example: cancer

outline the procedure for Sanger Dideoxy sequencing of DNA

a method to obtain the base pair sequence of a piece of DNA using acrylamide gel separation with P32 labeled nucleotides Process: primer binds to complementary site of template strand being sequenced DNA polymerase synthesizes a new strand by extending the primer complementary nucleotides are incorporated incorporation of deoxynucleotide results in continued synthesis incorporation of labeled deoxynucleotide results in strand termination and a labeled DNA fragment separated by size (small go farther) the length of DNA sequence and the color of the fragment represent the position of the base relative to the primer and the type of base (A,C,G,T)

describe SNP chips

a microarray that detects single base pair changes, polymorphisms and point mutations process: labeling of genomic DNA fragments that hybridize to DNA spots on a a microarray slide: each spot on the slide represents various alleles of a gene array is scanned to visualize where the gene spots hybridized advantage: thousands of probes (spots) may be searched in a single experiment Example: may detect variations in the CYP450, Prader Willi

describe array comparative genomic hybridization (CGH)

a molecular cytogenetic technique that allows detection of DNA sequence copy number changes throughout the genome in a single hybridization detects changes associated with 1Mb of DNA: 3,000 spots for genome maps locations (see slide 57) USE: to compare patient to normal DNA detects genome amplifications and deletions; TUMORS Limitations: does not detect balanced rearrangement EXAMPLE: Trisomy 18 Other Considerations: what defines normal copy number variants? what to do with unknown significance (would you tell a patient if they had a variable gene that may or may not have significance?)

what are regulatory mutations

alteration of promoter sequence

define amplification of repeat sequence mutations

amplification of repeat sequence causes genomic instability a transposon may disrupt gene

Principle of hybridization

annealing of complementary ssDNA

chromosomal analysis of gains, losses and amplifications of DNA

array CGH

what analysis identifies mapping of chromosomal breakpoints

array CGH

differentiate between single gene techniques (northern blot, ASO, RFLP and microarray techniques)

arrays analyze many genes southern blot, northern blot, western blot, PCR, ASO, and ASO PCR all analyze one gene single gene analysis is useful for confirming an array ex, Northern blot would be used to confirm a gene overexpressed by a cDNA microarray

describe cDNA microarrays

cDNA is made from RNA DNA is easier to work with, and cDNA only shows genes associated with products (excludes junk DNA, introns, etc.)

describe protein arrays

cDNA to identify genes expressed in a tissue USE: for comparative analysis, cancer Types: antibody, antigen, protein-drug interactions, protein-protein interactions, enzyme-substrate interactions, ligand arrays, carbohydrate chips

what are the limitations of PCR

carriers are often undetected solved by MLPA

describe FISH

fluorescently labeled probes used to hybridize partially denatured chromosome spreads Considerations: probes are 10-100 kb or longer improved resolution over G-banding facilitate the automation of genetic analysis EXAMPLES: 22q11 (DiGeorge), microdeletion on the X-chromosome in Steroid Sulfatase Deficiency, Trisomy-21 Limitations: must know what to look for Types: Chromosome-specific unique sequence probes or Gene-specific probes: uses probes derived from specific gene USES: identify submicroscopic deletions, translocations, and duplications of genes SKY or M-FISH: uses whole chromosome specific probes labeled with unique colored fluorescent dyes specific to each chromosome USES: characterizing complex chromosomal rearrangements & identifying the chromosomal origin of rearranged genetic material; ex, cancer

explain the circumstances that dictate whether direct or indirect molecular diagnosis can be used

if chromosomal location of a disease gene is known, but the gene itself has not been identified, DNA diagnosis can be performed with polymorphic DNA markers: this is called indirect analysis. when the gene is identified and the types of mutations are limited, direct methods for mutation detection may be possible.

describe how recombination applies to linkage analysis

if two loci are close together on a chromosome, the chances of recombination are low, these are called linked loci and may be useful for identification of mutant genes conversely, recombination may result in a diseased individual without the marker or result in a non-affected individual with the marker bottom line: recombination reduces the chances of finding a marker to associate with a particular diseased gene

differentiate between indirect and direct methods of mutation detection

indirect methods for mutation detection rely on two or more related persons with the disease to identify markers and track the disease in other family members direct methods are analysis of specific dna regions known to cause the disease

describe insertion/deletion (in-frame, frameshift) mutations

inframe: entire codon lost but does not change the rest of the sequence (pg 97) out of frame/frameshift: all subsequent aa codes are changed. likely frameshift outcomes: early in gene: stop codon created late in gene: extension of protein

what chromosomal analysis identifies aneuploidy

karyotype G-banding FISH

differentiate between techniques that are able to identify whole chromosome defects (aneuploidy) and chromosomal microdeletions

karyotyping can identify whole chromosome defects, such as Trisomy 21, while microdeletions are detected by FISH and Array CGH

Linkage analysis

linkage analysis has two main uses: 1) mutation is not known 1) disease exhibits allelic heterogeneity the gene of interest must be linked to a known marker locus the marker is analyzed in all affected persons in the family: if the marker is present in all the affected members, the marker may be linked to the disease causing gene the maker should be absent in all persons who are not affected identification of the disease causing gene is accomplished by sequencing nearby regions of the genome (now we can use the human genome databases)

automated DNA sequencing

loads all four reaction products into single lanes of the electrophoresis gel and capture of sequence data during the electrophoresis run four separate dyes are used as labels for the base-specific reactions a laser beam causes the dye to fluoresce need to be able to read for test! (pg70)

next generation sequencing

massively parallel genomic sequencing single stranded DNA segments to be sequenced are hybridized to primer sequences and attached to microarray chip

triple repeat expansions

may lead to gain of function

discuss DNA fingerprinting

only discussed in the context of it is getting cheaper!

describe substitution (missense, nonsense) mutations

point mutations of one base pair cause: silent mutations: does not change aa code missense mutations: changes protein nonsense mutation: creates stop codon

transition mutation

pyrimidine to pyrimidine (T and C switch) purine to purine (A and G switch) Example:Hemochromatosis (C to T)

what are the requirements for performing linkage analysis

requirements: 1) large family with several affect individuals 2) linkage map (markers near region of interest)

RFLP

restriction fragment length polymorphism is a direct detection method that uses a restriction endonuclease on a PCR with subsequent electrophoresis to identify deletions or mutations that resulted in a loss or gain of a restriction site only palindromes are recognized by restriction enzymes! EXAMPLE, sickle cell anemia considerations: in comparing DNA from two unrelated persons, there is a single bp difference about every 1,000bp: this may affect a restriction site, resulting in different patterns of DNA fragments 2) probe position may affect banding pattern 3) PCR more commonly used with RFLP than southern blot

Allele Specific Oligonucleotide Probes (ASO)

short oligonucleotide sequences that specifically bind to a single allele of a gene 1) exact gene mutation must be known! 2) not useful for allelic or genetic heterogeneity EXAMPLES, cystic fibrosis, hemochromatosis

why is the choice of restriction enzyme important in analyzing sickle cell anemia by RFLP

sickle cell anemia mutation creates one restriction site and destroys another (see slide 18)

describe mutations of splice-site

splice donor mutation: intron to be included in the mRNA > bigger OR may shorten protein product if a stop codon is in the intron splice acceptor mutation: may lead to loss of exon & a frame shift

what is automated cytogenetics

used with FISH analysis allows digital chromosome identification, sorting, and detection of abnormalities associated with translocations, deletions and duplications

describe a DNA microarray

useful for analyzing microdeletions and microduplications has 3000 spots of 1MB chunks