RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids - PowerPoint PPT Presentation

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RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids
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RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids

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  1. RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids - DNA Fingerprinting: forensic science, paternity/materity test, genetic testing for inherited disease - gene expression profiling/mutation analysis for identification of disease associated genetic alteration Lecture 2: Protein - from protein biochemistry to drug development

  2. Molecular Biology Techniques: Lecture 1 - Nucleic Acids Introduction to DNA (1) chemical structure (2) chromsome structure (3) genetic information - species/individual diversity (4) transcription/translation - from DNA -> RNA -> protein B. DNA Fingerprinting/Gene Expression Profiling (1) basis/rationale (2) Methods - Restriction Endonuclease - Agarose Gel Electrophoresis - Southern Blotting - Polymerase Chain Reaction (PCR) - DNA Sequencing - Northern Blotting - Microarray

  3. DNA • Deoxyribonucleic acid • composed of a chain of nucleotides • Each nucleotide contains: • (1) phosphate group • (2) five carbon sugar (blue) • (3) flat aromatic ring (base, orange) • There are 4 bases: • adenine (A), • thymine (T), • cytosine (C), • guanine (G) • A & G are purine (double-ringed) • T & C are pyrimidine (single-ringed) http://www.nvo.com/jin/nss-folder/scrapbookcell/4%20nucleotide.jpg

  4. DNA • DNA = nuleotide chain • linked by phosphodiester bond • between 5’ phosphate group • of one nucleotide to 3’ hydroxyl • group of the adjacent nucleotide • in cells, DNA exist as • double stranded helix. • Watson-Crick Base Pairing: • A - T • G - C http://www.mcat45.com/images/Nucleotide-bases-mcat.png

  5. Why packing? -nucleus ~6um (6x10-6m) in diameter -total length of DNA in human genome ~1.8m (6 ft long!)

  6. DNA stores genetic information essential for the development of an organism “Central Dogma” (Francis Crick, 1956): transcription protein DNA RNA translation replication

  7. DNA stores genetic information essential for the development of an organism Phylogenetic tree

  8. Monkey DNA = Homer DNA

  9. Homer DNA = Marge DNA Genetic Polymorphisms

  10. DNA polymorphism • “alternative form of chromosome content” • Coding region: • (1) nucleotide sequence difference without changing • amino acid sequence

  11. 20 amino acids, 64 possible three-base codons

  12. DNA polymorphism • “alternative form of chromosome content” • Coding region: • (1) difference in the nucleotide sequence without • changing the amino acid sequence • (2) difference in the nucleotide sequence with a • change in the amino acid sequence • (Genetic polymorphism could lead to disease • predisposition) • Noncoding region: • (1) difference in the nucleotide sequence • (2) difference in number of nucleotide repeat units

  13. VNTR = Variable Number Tandem Repeats • small nucleotide repeats • ~20-100 base pairs per • repeat • number of repeats varies • among unrelated individuals

  14. Forensic DNA analysis/DNA Fingerprinting/DNA Profiling • - finding the criminal by • placing a suspect at a • crime scene • paternity/maternity test • genetic testing for • inherited disease

  15. DNA Fingerprinting Methods RFLP Analysis PCR Analysis

  16. RFLP Analysis = Restriction Fragment Length Polymophism Techniques involved: Restriction Enzyme Digestion Agarose Gel Electrophoresis Southern hybridization

  17. Digestion of DNA Restriction enzyme = DNA nuclease that cuts double stranded DNA at a specific sequence (4-10bp)

  18. Restriction Mapping • - a piece of DNA may have multiple cutting sites by a • particular restriction enzyme • by analyzing the sizes of the digested DNA fragments, • one can create a restriction map • useful to confirm/identify the identity of a piece of DNA

  19. Agarose Gel Electrophoresis How to analyze DNA restriction fragments?

  20. Principles of Electrophoresis DNA is highly negatively charged

  21. Results of Electrophoresis

  22. DNA polymorphism may result in different restriction digest patterns among different individuals at certain gene locus: Individual 1 Individual 2

  23. DNA profiling for inherited disease

  24. DNA profiling for inherited disease visualization of the digested genomic DNA on agarose gel - Too many bands!

  25. Southern Blot (DNA)

  26. DNA profiling for inherited disease

  27. Variable Restriction Fragment Lengths Containing VNTRs

  28. DNA Fingerprinting Methods RFLP Analysis Polymerase Chiain Reaction (PCR) Analysis Advantage: requires much less DNA materials

  29. PCR developed by Dr. Kary Banks Mullis in 1983 • a technique to amplify a piece of DNA exponentially in test tube • required a heat stable DNA polymerase (Taq) and two oligo primers 25 cycle -> up to 1.7x107 amplification

  30. DNA profiling for criminal investigation 1 2 3 4 5 6 DNA obtained from the crime scene DNA obtained from 6 individual suspects http://en.wikipedia.org/wiki/Image:D1S80Demo.gif

  31. How to identify disease related mutation?

  32. Mutation analysis If you know what gene to analyze… If you have no idea what gene to go after…. PCR amplified your gene of interest from genomic DNA isolated from normal individual and disease carrier Comparative Genome Hybridization (detect genome wide gain/loss of DNA content) DNA sequencing single-stranded conformation polymorphism

  33. DNA sequencing • Maxim-Gilbert method • (old method, very toxic) • -dideoxynucleotide method

  34. Maxim-Gilbert Sequencing

  35. Dideoxynucleotide sequencing DNA synthesis requires 3’ OH group

  36. Single-stranded conformation polymorphism (SSCP) • single-stranded DNA forms • secondary structure • secondary structure may be • altered by a single base • substitution • -mutation(s) can be detected at • 50-100% rate within <200bp • PCR fragment Gasser et al., 2007

  37. Comparative Genome Hybridization array based metaphase chromosomes

  38. What is the consequence of the mutation? • (1) Gene expression • mutation at promoter • mutation at splice site? Transcription and Splicing DNA Exon 3 Exon 1 Exon 2 intron intron intron intron hRNA mRNA

  39. To analyze RNA transcript- Northern Blot • RNA, rather than DNA is blotted • Same techniques as Southern blot • Can determine levels of RNA expression in tissues, cell types etc. • Allows detection of any abnormal size of the transcript

  40. Northern Blot occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/Northernblot.html

  41. Microarray - global gene expression profiling Each spot on the array chip represents one gene

  42. Reverse Transcription

  43. To confirm or reproduce a disease-associated phenotype due to the loss of expression of the gene of interest: Gene knockout (2) RNA interference

  44. Gene Knockout http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig4-16.jpg

  45. RNA interference Dicer = RNAse III RISC = RNA - inducing silencing complex

  46. Protein “Central Dogma” (Francis Crick, 1956): transcription protein DNA RNA translation replication

  47. Protein Synthesis Overview Catalyzed by Ribosome tRNA Met initiates at P site Correct tRNA binds at A site

  48. To study the biochemical activity of the protein of interest… Recombinant Protein Production