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9-1-05 Molecular medicine


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A DNA sequence variation may or may not be pathogenic
an allele present in more than 1% of the general population
G2983>A means what?
a nucleotide exchange of guanine for adenine at position 2983
What does C282Y mean?
a missense mutation C is original amino acid, the position, and the new amino acid (tyrosin)
What does W1282X mean?
nonsense mutation original, position, folowed by X
what does 711+1G->T
mutation in intron/splicing site 1 is the 5' donor splice site
What is a point mutation?
a single base pair change in DNA
What is missense mutation?
changes in amino acid.
What is silent mutation?
does not change Amino acid
what is conservative?
changes to an amino acid with simliar properties
What is a nonsense mutation?
creates a stop codon Very deletarious to the protein!
What are RNA splicing mutations?
they disrupt the consensus sequences from splicing, or create alternative sites (pathogenic)
What are small insertions/deletions?
Will change reading frame (frameshift) if not in multiples of 3. (pathogenic)
What is loss of function?
reduces or eliminates the amount of funstional protein. ie. CF
What is gain of function?
increases production of a normal protein ie Anchondroplasia
What are novel property mutations?
changes function of the protein ie sickle cell anemia no effect on O2 transport, Huntington disease
What is dominant negative?
An allele that disrupts the function of the wild type allele in the same cell. Its worse than having none. Osteogenesis imperfecta.
What are triplet repeat expansions?
unstable mutations in which have expansion of a segment of DNA that contains 3 nucleotides
-lead to abnormalities of gene expressionandfunction
-repeats can be anywhere in gene even in intron
Huntington, fragile X, spinal cerebellar ataxis, Freidreichs ataxias, myotonic dystrophy
What is anticipation?
degree of expressivity or penetrance changes, usually increases, from one generation to the next
What is parental bias?
tenedency for a repeat to expand differs from a male vs. female
important concepts of mitochondrial genetics
-own genome
high mutation rate
-maternal origin
-variable inheritance and expression (diff phenotypes even in same family)
-heteroplasmy (diff tissue have diff levels)
-segregate as mendelian traits
Couple key ideas about Real time PCR
very rapid and sensitive 1/100,000
-use melting point analysis mutation destablizes and reduces melting point. Heterozygotes have 2 Tm's.
What is RFLP?
Restriction fragment length polymorphism use restriction enzymes to cut DNA at specific sites palindromic sequences
-good if mutation adds or deletes a site so get change in pattern
What are "ARMS"?
-Allele specific PCR amplification
-good if mutation doesnt change restiction site
-faster than PCR RFLP
-design primers specific to mildtype and mutated sequence
-needs 3' end to add and wildtype primer will extend
-score for absence/presence of a band)
What is ASO or Dot Blot?
-allele specificoligonucleotide hybridization
-short probes if perfect match will hybridize
-useful if no informative restriction site
-if probe binds you get color
-can be adapted for high throughput by spotting oliogos and probing patients labelled DNA (reverse dot blot)
What is microarray?
ASO for known mutations probes can be spotted onto chip, fluoresce labeled patient DNA will bind to mutant or WT or both. Mismatches wont bind.
Southern Blotting +/-/
can see large scale duplications and deletions expansions
-disadvantages: takes forever , expensive
DNA sequencing
incorporates random ddNTPs that dont allow pol to extend
+speels out DNA seq (gold standard)
-expensive laborious
Newer DNA sequencing
automated, uses fluorescence read by laser/CCD camera
should be 1 peak/position.
overlapping (go over)
What are microsatellites?
short tandem repeats
-zygosity testing
-maternal cell conamination
-UPD testing
What is locus heterogeneity?
more than one locus associated with a specific clinical phenotype (lots genes, 1 disease)
Modifier gene
gene that alters phenotype associated with mutations in a non allelic gene (thalassemia and sickle cell interaction)
Clinical or Phenotype heterogeneity
association of more than one phentype with mutations at a single locus. 1 gene, variable disease CF)
Allelic heterogeneity
multiple alleles at a locus ( many possible mutations in 1 gene, CF)
Diagnostic Testing
having a diagnosis is important to know recurrence risk, access support groups, avoid other invasive $$ procedures
Predictive testing
asymptomatic patients want to know. Counseling
Carrier testing
most informative when familial mutation is known disouraged in children
Prenatal testing
familys specific mutation should be known before testing offered
______ is used for biochemical disorder testing
enzyme assay
When family history of a disease, should______________
then subsequent at risk people can have very informative test for only that mutation
Affected person tested

CF carrier screening,ehthnicity matters!

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