Molecular Diagnostics for Dummies

1. Molecular Diagnostics for Dummies Capt G. Shane Hendricks, USAF, BSC, MS, MT (ASCP) Associate Chief, Diagnostic Services 59th Laboratory Squadron

2. Foreword • Brief history and basic molecular genetics concepts • Mendel to Watson-Crick • Central Dogma • Molecular techniques - clinical and research • PCR • REs/Gels/RMs • Blots • Etc. • Example applications within clinical laboratory

3. Brief History and Basics

5. History of Genetics • Mendelian Genetics • Gregor Mendel - 1863 • Crossed pea plants w/ varied traits • Proved trait inheritance • What was the biochemical basis of traits? • 4 properties apparent: copied, stored, expressed, changed • Prior to 1944 - protein • More abundant • DNA thought too simple

6. History of Genetics • DNA first isolated in 1869 - Friedrich Miescher • Called material “nuclein” (came from nucleus) • Phoebus Levene in 1919 • Identified nucleotide units • Base, sugar, phosphate components • Believed repeating; too simple for genetic information • ATGC • First proof of genetic material - transformation • 1928 - Griffith

7. Griffith

8. What was transforming factor? • Avery, McLeod, McCarty - 1944 • Used “cell-free” system • Factor was called DNA

9. Contribution of Viruses • 1952 - Hershey, Chase • Used bacteriophages • Used radioactive isotopes of Phosphorus/Sulfur

10. What about Eukaryotes? • Indirect evidence • Haploid cells - 1/2 material of diploid cells • Mutagenesis - 260 nm mutate material, not protein • Proteins absorb at 280 nm • Direct evidence • Transfection - material introduced into cells; new trait acquisition

12. Structure of DNA • Many genetic pioneers found puzzle pieces • William Astbury - 1937 • Rosalind Franklin - 1951 • James Watson, Francis Crick - 1953 • Put pieces together! • Built first accurate model

13. Structure of DNA (cont.) • Two polynucleotide chains in double helix • Each chain associates by hydrogen bonding • A:T (2 bonds), G:C (3 bonds) • Chains run anti-parallel • Sense - equivalent to mRNA transcript • Anti-sense - “compliments” Sense strand • Sugar-phosphate backbone outside • Bases lie flat inside, perpendicular to central axis • Grooves

14. Structure of DNA (cont.)

15. nucleotide ssDNA A } T Nucleoside G C NTP = ATP, GTP, etc. Nucleobases: Purines pyrimidines A

16. dsDNA complementation

17. Nuts and Bolts • Biomolecular polymers and information • Nucleic acids (DNA and RNA) store information • Quaternary variation 2n 4n

18. Nuts and Bolts (cont.) • Stored information transcribed first • Transcript next translated into peptides • Oligopeptides  polypeptides protein • Variety of structural and/or catalytic functions

19. Central Dogma ???

20. More Basics (cont.) • Gene - discrete sequence • Genome - all genetic information • Expression • Regulation - off or on? • Promoters - upstream • Complex in eukaryotes • TATAAA • Regulation-associated pathology • Asthma • Beta thalassemia • Various cancers • Chimeric proteins - e.g., Philadelphia Chromosome (CML)

21. Eukaryotic Chromosomes Charges? Chromatid

22. Eukaryotes (cont.)

23. Eukaryotes (cont.) P p • Haploid vs. Diploid • Homo- vs. Heterozygous • Dominant and Recessive • Phenotype vs. Genotype

24. Prokaryotic Chromosomes

25. Prokaryotes (cont.)

26. Prokaryotes (cont.)

27. Replication

29. Replication • Mitosis, meiosis • Faithful DNA reproduction • Theories • Dispersive • Conservative • Semi-conservative

30. Replication (cont.)