Download Molecular Diagnostics for Dummies

59th Medical Wing
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Molecular Diagnostics for Dummies
Capt G. Shane Hendricks, USAF, BSC, MS, MT (ASCP)
Associate Chief, Diagnostic Services
59th Laboratory Squadron
Foreword
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Brief history and basic molecular genetics concepts
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Mendel to Watson-Crick
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Central Dogma
Molecular techniques - clinical and research
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PCR
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REs/Gels/RMs
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Blots
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Etc.
Example applications within clinical laboratory
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Brief History and
Basics
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History of Genetics
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Mendelian Genetics
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Gregor Mendel - 1863
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Crossed pea plants w/ varied traits
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Proved trait inheritance
What was the biochemical basis of traits?
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4 properties apparent: copied, stored, expressed, changed
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Prior to 1944 - protein
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More abundant
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DNA thought too simple
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History of Genetics
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DNA first isolated in 1869 - Friedrich Miescher
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Called material “nuclein” (came from nucleus)
Phoebus Levene in 1919
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Identified nucleotide units
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Base, sugar, phosphate components
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Believed repeating; too simple for genetic information
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ATGC
First proof of genetic material - transformation
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1928 - Griffith
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Griffith
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What was transforming factor?
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Avery, McLeod, McCarty - 1944
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Used “cell-free” system
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Factor was called DNA
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Contribution of Viruses
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1952 - Hershey, Chase
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Used bacteriophages
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Used radioactive isotopes
of Phosphorus/Sulfur
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What about Eukaryotes?
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Indirect evidence
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Haploid cells - 1/2 material of
diploid cells
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Mutagenesis - 260 nm mutate
material, not protein
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Proteins absorb at 280 nm
Direct evidence
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Transfection - material
introduced into cells; new
trait acquisition
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Structure of DNA
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Many genetic pioneers
found puzzle pieces
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William Astbury - 1937
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Rosalind Franklin - 1951
James Watson, Francis
Crick - 1953
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Put pieces together!
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Built first accurate
model
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Structure of DNA (cont.)
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Two polynucleotide chains in double helix
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Each chain associates by hydrogen bonding
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A:T (2 bonds), G:C (3 bonds)
Chains run anti-parallel
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Sense - equivalent to mRNA transcript
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Anti-sense - “compliments” Sense strand
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Sugar-phosphate backbone outside
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Bases lie flat inside, perpendicular to central axis
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Grooves
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Structure of DNA (cont.)
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NTP =
ATP, GTP,
etc.
Nucleobases:
Purines
pyrimidines
Nuts and Bolts
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Biomolecular polymers and information
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Nucleic acids (DNA and RNA) store information
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Quaternary variation
n
2
4
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Nuts and Bolts (cont.)
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Stored information transcribed first
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Transcript next translated into peptides
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Oligopeptides  polypeptides  protein
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Variety of structural and/or catalytic functions
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???
Central
Dogma
More Basics (cont.)
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Gene - discrete sequence
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Genome - all genetic information
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Expression
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Regulation - off or on?
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Promoters - upstream
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Complex in eukaryotes
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TATAAA
Regulation-associated pathology
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Asthma
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Beta thalassemia
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Various cancers
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Chimeric proteins - e.g., Philadelphia Chromosome (CML)
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Eukaryotic Chromosomes
Chromatid
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Eukaryotes (cont.)
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Eukaryotes (cont.)
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Haploid vs. Diploid
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Homo- vs. Heterozygous
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Dominant and Recessive
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Phenotype vs. Genotype
P
p
P
PP
Pp
p
Pp
pp
P
p
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Prokaryotic Chromosomes
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Prokaryotes (cont.)
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Prokaryotes (cont.)
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Replication
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Replication
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Mitosis, meiosis
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Faithful DNA
reproduction
Theories
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Dispersive
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Conservative
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Semi-conservative
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Replication (cont.)