Molecular Medicine Flashcards

1
Q

DNA sequencing is most useful when?

A

—when the exact nature of the disease-causing mutation is known

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2
Q

Indirect Detection and Potential Problems w/ it

A
  • -if exact mutation is not known one looks for presence of a linked marker
  • -problem: marker may have separated from disease allele by recombination (if 10% recombination between marker and gene then one can only be 90% sure that a child without the marker is disease free)
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3
Q

Direction Detection and Potential Problems

A
  • -if exact mutation is known, one can test Pt. DNA for it (basically sequence the DNA directly)
  • -doesnt work if you dont know what mutation is since lots of SNP’s could show up as well
  • -problem: Pt. might have a novel, rare or unknown mutation that is not detected
  • -low penetrance means child w/ mutation might be healthy
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4
Q

What to sequence when considering sequencing?

A
  • -not whole genome (not useful for clinical practice)
  • -exome: all genome that is expressed 30 mil. bps
  • -SNP typing: fewer than 10 mil. SNP’s in genome so to learn about individual characteristics of genome do SNP typing
  • -only a few hundred SNP’s have been associated w/ disease and health risk
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5
Q

Copy Number Variations

A
  • -presence of small chromosomal insertions or deletions

- -most are benign polymorphisms

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6
Q

Comparative Genome Hybridization

A
  • -used to detect unbalanced chromosomal abnormalities
  • -Pt. and control single stranded DNA labeled w/ flourescent dye and then hybridized to metaphase chromos to see how they match up
  • -CNV evident by uneven labeling of the template chromsome i.e. excess of Pt. label fluorescence indicates a duplication in Pts. genome
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7
Q

FISH

A
  • -fluorescence in-situ hybridization
    • allows identification of specific chormosomal locus on a metaphase chromo by hybridization w/ a specific fluorescent probe
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8
Q

Hybridization of Nucleic Acids

A
  • -denature Pt. DNA and attach to membrane

- -expose to bunch of labeled complimentary sequences and see what hybridizes

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9
Q

How is PCR used to detect genetic polymorphisms and mutations?

A
  • -mutations can be detected w/ oligonucleotides that exlcusivley bind to either mutant or wildtype alleles (using mutant specific ASO’s as primers, only mutant alleles will be amplified)
  • -Insertions or deletions between primer binding sites become obivous since you will get strands of different lengths amplified
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10
Q

Real-Time PCR

A
  • -used for DNA quantification
  • -amount of DNA amplified is measure at end of each cycle
  • -determined how many cycles are required to reach a threshold
  • –higher the number of template molecules = the fewer cycles needed to reach threshold
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11
Q

Reverse Transcriptase PCR

A
  • -RNA —> DNA (by reverse transcriptase)
  • -this cDNA serves as template for PCR
  • -the more RNA there is = the more DNA will be amplified
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12
Q

Allele Specific Oligonucleotides (ASO’s) Tool

A
  • -used to detect SNP’s and mutations
  • -requires detailed knowledge about nucleotide sequence of gene under study
  • -apply ASO spots for WT and mutant to slide
  • -amplify Pt. DNA and label and hybridize to ASO spots
  • -if both spots are labeled then Pt. is heterozygous and has both alleles
  • -very specific method, ASO primers wont bind if one base is wrong
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13
Q

DNA microarrays

A

–basically just the ASO technique multiplied out and can be used for genetic screening of a Pt. for hundreds of known disease causing mutations

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14
Q

Analysis of gene expression by microarray

A
  • -mRNA from Pt. and control is taken and reverse transcribed to ssDNA and labeled (Pt. red, control green)
  • -then hybridized to microarray plate w/ oligonucleotides on them
  • -DNA’s compete for binding
  • -if certain gene is not expressed in a sample then there will be no ssDNA that will bind to that oligo and only the control DNA (green will bind)
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15
Q

Pharmacogenomics and polymorphism importance

A
  • -polymorphisms in genes coding for drug targets determine how effective a drug inhibits the target enzyme
  • -polymorphisms in drug metabolizing enzymes determine how fast a drug is cleared from system
  • -goal is to find polymorphims that affect drug response in order to work out individual treatment plan
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16
Q

Prenatal Diagnosis Techniques

A
  • -amniocentesis (gold standard): amniotic fluid is withdrawn-fluid contains fetal skin cells to be analyzed (15-16 weeks gestation)
  • -chorionic villus sampling: cells taken from chorion (10-12 weeks gestation) cells are extraembryonic in origin and could possible miss a mosaicism
17
Q

Direct Sequencing used to detect?

A

–point mutations and SNPs

18
Q

Whole Exome Sequencing used to detect?

A

–point mutations/ SNP’s in EXONs –not introns or intergenic regions

19
Q

SNP typing used to detect?

A

–SNPs in entire genome

20
Q

PCR used to detect?

A

–small insertions or deletions

21
Q

Reverse transcriptase PCR used to detect?

A
  • -expression levels for small number of genes (8-12)

- -use RNA

22
Q

Allele Specific Oligonucleotides used to detect?

A

– point mutations, small insertions and deletions

23
Q

Gene expression array used to detect?

A
  • -uses RNA

- -expression levels of thousands of genes-not intergenic regions or introns

24
Q

Methylated DNA used to detect?

A

–epigenetic changes, DNA methylation

25
Q

Comparative Genome Hybridization used to detect?

A
  • -insertions, deletions, and aneuploidies in the kilo- to megabase range
  • -CNV’s
26
Q

FISH used to detect?

A

–copy number of a selected chromosomal region

27
Q

ELISHA used to detect?

A

–amounts of protein in sample

28
Q

Western Blot used to detect?

A

–amounts and size of protein in sample