Genetics-Grody

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Prenatal diagnosis sources

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1)amniocentesis
2) chorionic villus samples (CVS)
3) embryonic blastomeres for preimplantation diagnosis (single cell PCR)
4) Fetal cells in maternal blood

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Collection for genetic testing

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any cell in human body

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Clinical purposes of molecular genetic tests

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-clinical diagnosis/confirmation
-carrier screening
-prenatal diagnosis
-newborn screen
-presymptomatic diagnosis/predisposition screening

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molecular genetics vs molecular oncology

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molecular genetics more concerned w/ inherited mutations

molecular oncology concerned w/ somatic mutations

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application of DNA testing

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-transplantation entrapment
-paternity testing
-forensic ID
-twin zygosity -mono vs. zygotic twins
-surgical mix-ups
-vet applications

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methods of analysis & interpretation

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-frequency of given matching allels multiply together = cumulative probablility

match = inclusion
mismatch = exclusion

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how do you detect & compare polymorphisms?

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Souther Blot-longer
VNTR=Variable Number Tandem
RFLP= Restriction Fragment Length Polymorphisms

PCR-shorter
STR=Short Tandem Repeats
SNP=Single Nucleotide Polymorphisms

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Types of DNA polymorphisms found in DNA fingerprinting

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 RFLP = Restriction Fragment Length Polymorphisms - restriction endonuclease recognition sites that are present in some people but not in others

 VNTR = Variable Number Tandem Repeats - strings of nucleotide sequences (usually of 16 or more nucleotides each), of different repeat number in different people, existing between fixed restriction endonuclease sites; also called minisatellites

 STR = Short Tandem Repeats - strings of oligonucleotide sequences that are shorter than VNTR (usually of 2, 3, 4, or 8 nucleotides each), of different repeat number in different people, not necessarily lying between restriction endonuclease sites; also called microsatellites

 SNP = Single Nucleotide Polymorphisms - single nucleotide differences between individuals that are not associated with restriction endonuclease sites

 HLA = the histocompatibility family of genes located on chromosome 6p; although not "junk" DNA, these genes are naturally so highly polymorphic that they can be used for identity studies at either the DNA level or using antibodies to distinguish varieties in the encoded proteins

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mitochonrial mutation

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-maternal inheritance
-heteroplasmy may require biopsy of affected tissue

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diseases of trinucleotide Repeat Expansions

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-fragile X
-huntingtons
-fredreich ataxia
-myotonic dystrophy
-spinocerebellar ataxia

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diseases where gene is known but not mutation

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-duchenne muscular dystrophy (2/3 due to large deletions
-cystic fibrosis (>1300 mutations)
-familial breast cancer
-multiple endocrine neoplasm

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diseases for which both disease & mutation are known

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-sickle cell anemia
-factor V leiden

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By order of diagnostic difficulty, the molecular classes of genetic diseases are

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1)both gene & causitive mutation known
2)gene known; mutation not
3)neither gene nor mutation known
4) caused by more than 1 gene interacting w/ enviornmental factors (polygenic & multifactorial)

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Unique features of molecular diagnostics of genetic diseases

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-examining individuals unique genotype
-test results have implications for blood reletives
-specimens from family members may be required (e.g., linkage testing)
-can predict disease before onset
-can predict disease before birth

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To detect many possible mutations in diseases w/ marked genetic heterogenity use:

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-PCR -several gene regions followed by sevel hybridization w/ panel of ASO probes

-Dot Blot -hybridization w/ pooled cocktail of ASO probes for several rare mutations at once

-PCR followed by reverse dot blot hybridization to an array of ASO probes bound to a paper strip or other solid support (e.g, cystic fibrosis)

-Manual or automated DNA sequencing (e.g., BRCA 1/2)

-Mutation scanning techniques (DGGE, SSCP, PTT)

-DNA chip

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To detect trinucleotide repeat expansions use:

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-sizing by PCR if expansion is of moderate length
-sizing by southern blot if expansion is of lare size ( too large for PCR)

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For large deletions use:

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-southern Blot
-fluorescence in sito hybridization (FISH)
-routine cytogenetics, if deletion is big enough to be visualized by light microscopy
-multiplex PCR (deletions of duchenne muscular distrophy)

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For pt mutations & microdeletions/microinsertions use:

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-southern blot or dot blot hybridization w/ ASO probes
-southern blot or PCR amplification, subjcting the products to digestion w/ particular restriction endonuclease whose natural cleavage sequence is either destroyed or created by mutation in question
- DNA sequence

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Utility of PCR

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-extremely sensitive
-large # of specimens
-not good for large regions of DNA

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genetic heterogenety

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many genetic & neoplastic diseases can be caused by more than 1 mutation, or more than one gene

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Types of mutations & results:

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-pt mutation- missense or nonsense coding, errors in RNA splicing

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Types of mutations & resutls:

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1)pt mutation - missense or nonsence coding, errors in RNA splicing
2) microdeletion - frameshift mutation
3)deletions- minus large portion of gene
4) trinucleotide repeat expansion
5) translocations - xchange b/n 2 chromosomes
6)rearrangement - shifting on same chromosome
7) amplifications - increase copy number of gene

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trinucleotide repeat expansion

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-tandem triplet repeat enlarges to greater than normal leanght w/in gene
-common in neuromuscular diseases

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Southern Blot

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a techniqe for probing gene sx involving endonuclease digestion of target DNA followed by gel electrophoresis, transfer of DNA fragment to a filter membrane, & hybridization w/ a DNA probe

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Predictive genetic testing

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analysis of mutation causing late onset, usually dominant diseases in presently healthy people

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polymorphism

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benign sequence change w/in DNA that is reletively common in the population

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point mutation

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a localized defect w/in a gene consisting of substitution of a single nucleotide or sometimes a small deletion or insertion

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PCR

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polymerase chain rxn - powerful technique for amplifying a specific region of a gene to obtain abundant purified DNA for further analysis

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mutation scanning

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any number of tecniques designed to detect presence of an unknown nucleotide change in gene, based on the resulting change in the genes physicochemical properties

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linkage analysis

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technique for tracking mutant gene w/n a family by deletion of polymorphisms located closely on same chromosome; required when direct mutation detection is not possilble because disease gene is unknown

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dot blot

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hybridization of DNA probe to a target specimen. Spotted onto a filter membrane

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DNA sequencing

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technique, manual or automated, delineating the order of nucleotides in gene or gene fragment

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DNA fingerprinting

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use of complex dna polymorphic patterns to ID & match individuals or specimens

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direct mutation testing

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detection of specific mutations in known disease genes

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deletion

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sizable defect in gene due to loss of a stretch of nucleotides

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ASO

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allele specific oligonucleotide- a short DNA probe that will specifically hybridize to either the normal gene or any particular gene sequence.