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Slide 1-

The Power of Molecular Biological Techniques Mark E. Sobel, MD, PhD Executive Officer American Society for Investigative Pathology mesobel@asip.org www.asip.org
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Overview Introduction to Molecular Pathology DNA, Restriction Enzymes, Hybridization, PCR Introduction to the Genome Applications to Molecular Medicine: SNPs and Chips
Slide 3-

TEST YOUR SCIENCE LITERACY Adapted from Dave Barry, Miami Herald Explain in your own words, what is DNA? DNA is deoxyribonucleicantidisestablishmentarianism, a complex string of syllables found inside your body in tiny genes called chromosomes. The information in your DNA determines your unique biological characteristics, such as eye color, Social Security number, and age. There is surprisingly little difference between DNA in humans, Democrats, and Republicans.
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Highly Sensitive and Specific by “Orders of Magnitude” from Powers of Ten, by Charles and Ray Eames
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BIOLOGY: THE STUDY OF LIFE WHOLE ORGANISMS ORGANS TISSUES CELLS INTRACELLULAR ORGANELLES CHEMICAL COMPONENTS
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CHEMICAL COMPONENTS OF LIFE PROTEINS LIPIDS NUCLEIC ACIDS: DNA RNA
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MOLECULAR BIOLOGY TECHNIQUES Molecular biology techniques utilize DNA, RNA, and enzymes that interact with nucleic acids to understand biology at a molecular level.
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MOLECULAR PATHOLOGY Molecular Pathology is a subspecialty of pathology that utilizes molecular biology techniques to: Detect normal and disease states (diagnosis) Predict disease progression (prognosis)
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SUBSPECIALTIES OF MOLECULAR PATHOLOGY INHERITED DISEASES (GENETICS) Cystic fibrosis Sickle cell anemia Predispositions to cancer INFECTIOUS DISEASES Bacteria Viruses Fungi
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SUBSPECIALTIES OF MOLECULAR PATHOLOGY HEMATOPATHOLOGY Leukemias Lymphomas SOLID TUMORS Breast cancer Colon cancer Brain cancer
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SUBSPECIALTIES OF MOLECULAR PATHOLOGY FORENSICS IDENTITY TESTING HLA parentage
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NUCLEIC ACIDS Genetic material of all known organisms DNA: deoxyribonucleic acid RNA: ribonucleic acid (e.g., some viruses) Consist of chemically linked sequences of nucleotides Nitrogenous base Pentose- 5-carbon sugar (ribose or deoxyribose) Phosphate group The sequence of bases provides the genetic information
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Bases Two types of bases Purines are fused five- and six-membered rings Adenine A DNA RNA Guanine G DNA RNA Pyrimidines are six-membered rings Cytosine C DNA RNA Thymine T DNA Uracil U RNA
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Base-pairing Hydrogen bonds are relatively weak bonds compared to covalent bonds Hydrogen bonds can form between a pyrimidine and a purine Watson-Crick base-pairing rules A T G C
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Hydrogen Bonds
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DNA: Helix In general, DNA is double-stranded. Double-stranded (ds) DNA takes the form of a right handed helix with approximately 10 base pairs per turn of the helix.
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Complementarity In the DNA double helix, purines and pyrimidines face each other The two polynucleotide chains in the double helix are connected by hydrogen bonds between the bases Watson-Crick base-pairing rules A T G C GC base pairs (bps)have more energy than AT bps Since one strand of DNA is complementary to the other, genetic material can be accurately reproduced; each strand serves as the template for the synthesis of the other
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Antiparallel Chains 5’p OH3’ Two strands of the DNA double helix are antiparallel and complementary to each other
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Gene A gene is a unit of inheritance Carries the information for a: -polypeptide -structural RNA molecule
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Nucleases
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Restriction enzymes Specific endonucleases Recognize specific short sequences of DNA and cleave the DNA at or near the recognition sequence Recognition sequences: usually 4 or 6 bases but there are some that are 5, 8, or longer Recognition sequences are palindromes Palindrome: sequence of DNA that is the same when one strand is read from left to right or the other strand is read from right to left– consists of adjacent inverted repeats
Slide 22-

Restriction enzymes (cont’d) Example of a palindrome: GAATTC CTTAAG Restriction enzymes are isolated from bacteria Derive names from the bacteria Genus- first letter capitalized Species- second and third letters (small case) Additional letters from “strains” Roman numeral designates different enzymes from the same bacterial strain, in numerical order of discovery Example: EcoRI E Escherichia Co coli R R strain I first enzyme discovered from Escherichia coli R
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Hybridization Nucleic acid hybridization is the formation of a duplex between two complementary sequences Intermolecular hybridization: between two polynucleotide chains which have complementary bases DNA-DNA DNA-RNA RNA-RNA Annealing is another term used to describe the hybridization of two complementary molecules
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Denaturation - Renaturation
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Probes Probe is a nucleic acid that can be labeled with a marker which allows identification and quantitation will hybridize to another nucleic acid on the basis of base complementarity Types of labels Radioactive (32P, 35S, 14C, 3H) Fluorescent FISH: fluorescent in situ hybridization chromosomes Biotinylated (avidin-streptavidin)
Slide 26-

Solid Support Hybridization Solid support hybridization: DNA or RNA is immobilized on an inert support so that self-annealing is prevented Bound sequences are available for hybridization with an added nucleic acid (probe). Filter hybridization is the most common application: Southern Blots Dot/Slot Blots Northern Blots In-silica hybridization (glass slides) in situ hybridization (tissue) Chromosomal (FISH) Microarrays
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Southern Blots Southern blotting is a procedure for transferring denatured DNA from an agarose gel to a solid support filter where it can be hybridized with a complementary nucleic acid probe The DNA is separated by size so that specific fragments can be identified Procedure: Restriction digest to make different sized fragments Agarose gel electrophoresis to separate by size Since only single strands bind to the filter, the DNA must be denatured. Denaturation to permit binding to the filter (NaOH) Transfer to filter paper (capillary flow) Hybridization to probe Visualization of probe
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Southern Blot Restriction enzyme
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Southern Blot
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Dot/Slot Blots DNA or RNA is bound directly to a solid support filter No size separation Ideal for multiple samples and quantitative measurements Important to establish specificity of conditions
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Slot Blot
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A Focus of Development: Automation User-Friendly, Faster, and Cost-Effective This electronic microarray is an example of "Lab-on-a-Chip" technology. It is an electrophoresis device that produces results up to 1000 times faster than conventional techniques while using much less sample.
Slide 34-

High Resolution Banding and FISH Control Signals Region-Specific Signal The chromosome banding technique performed 20 years ago missed the small deletion. High resolution banding developed more recently can elucidate the abnormality. Fluorescence in situ Hybridization (FISH) is a powerful technique in that it can reveal submicroscopic abnormalities even in non-dividing cells.
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Polymerase chain reaction PCR is the in vitro enzymatic synthesis and amplification of specific DNA sequences Can amplify one molecule of DNA into billions of copies in a few hours
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Applications of PCR Detection of chromosomal translocations Amplification across a translocation sequence Chromosome painting Detection of residual disease Infectious disease Forensics HLA typing Detection of Loss of Suppressor Genes Loss of Heterozygosity (LOH)
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Genome Literacy Genome: The entire DNA of an organism Humans diploid (chromosome pairs) 6 x 109 bp per diploid genome Haploid genome is one set of chromosomes Chromosome: structure found within a cell nucleus consisting of a continuous length of ds DNA Humans 22 pairs of autosomal chromosomes 2 sex chromosomes
Slide 38-

Human Genome Project 40,000 genes Speaking a language of molecular fingerprints Gene expression is another language of complexity
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Genome Mapping Terms Locus: a position on a chromosome Allele: alternate form of DNA at a specific locus on the chromosome Each individual inherits two copies of DNA Maternal Paternal Homozygous alleles: the two copies are identical Heterozygous alleles: the two copies are different
Slide 40-

Restriction fragment length polymorphism RFLP is a polymorphic allele identified by the presence or absence of a specific restriction endonuclease recognition site: GAATTC versus GATTTC RFLP is usually identified by digestion of genomic DNA with specific restriction enzymes followed by Southern blotting Regions of DNA with polymorphisms: Introns Flanking sequences Exons
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Genetic Variation Most genes have small sequence differences between individuals Occur every 1350 bp on average Some of these polymorphisms may affect: How well the protein works How the protein interacts with another protein or substrate The different gene forms containing polymorphisms are called alleles
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Mutation detection Sequence DNA Hybridization Methods Blotting Chips Restriction enzyme polymorphisms: GAATTC versus GATTTC SNPs (single nucleotide polymorphisms)
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SNPs Single nucleotide polymorphisms Distinction from mutations
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ASO Allele Specific Oligonucleotides ATGTGGCCATGTGGC ATGCGGCCATGTGGC ASOs can be used to detect SNPs (single nucleotide polymorphisms)
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More About SNPs SNPs in exons are called coding SNPs SNPs in introns or regulatory regions may affect transcription, translation, RNA stability, RNA splicing
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Pharmacogenomics Cytochrome P450 Uptake and metabolsim of drugs Seizure disorders Psychiatric disorders Cancer therapy
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Resources www.amptestdirectory.org is an online directory of laboratories that perform molecular techniques. www.genetests.org has an illustrated glossary and good explanations of genetic testing. http://www.ornl.gov/sci/techresources/Human_Genome/education/images.shtml has links to many educational resources and images. http://www.dnalc.org/resources/resources.html has an animated DNA primer targeted at the level of a “bright teenager.” It is a part of the website of the Dolan DNA Learning Center of Cold Spring Harbor Laboratory.
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aRA r = 0.91 log10(ratio), T3-3 Microarrays FISH Laser microscope genome Tissue arrays