Genetics 15 and 16 Recombinant DNA

Name Fluorescence in situ hybridization (FISH) Description Detection of chromosome aberrations Can do spectral karyotyping o Might be able to detect double minute chromosomes Concepts utilized: o Unique DNA seq has unique complementary seq o Base pairing b/w complementary strands is reversible Reconstitution of dsDNA from ssDNA = hybridization Method: o DNA seq present in chromosome fixed on slide Denatures to ssDNA o Fluorescently labeled DNA seq (probe DNA) prepared in vitro Denatured to ssDNA by heat or alkali treatment Probes are 100s and 1000 bp in length o ssDNA probe hydridize to ssDNA sample Detect: o Increases or decreases in chromosome number (aneuploidies, duplications) o Deletions and translocations of large chr segments Adv of FISH: o Dont need high quality metaphase chromosome prep o Interphase nuclei can be used o Specificity of hybridization allow rapid determination of identity of rearranged chromosome material or fragments Limitation: o Low resolution = Mutations caused by changes in one or a few nucleotides wont be detected Cant detect missense, nonsense mutations or small deletions/insertions Probes are cDNA fragments o Usually amplified by PCR o cDNA are copies of transcribed seq in cells mRNA in cell types are distinct Method: o Probes deposited on solid support, usually glass slide o Samples (normally poly(A)+RNA) are labeled using fluorescent dyes o At least 2 samples hybridized to chip Eg. tumor cell relative to normal cell o Fluorescence at diff wavelengths measured by scanner Principle clinical application: o Classifying neoplastic disease by differential gene exp profiles Graded tumors based on microarrays Biomarkers for resistance to chemotherapy Herceptin = antibody used to treat aggressive late-stage breast cancer Herceptin resistance a major prob Reduced PTEN exp enhances Herceptin resistance Expression profiling of breast tumor tissue enhances timely application of appropriate therapy o Diagnostic markers for infectious disease Permits amp of specific DNA seq from complex heterogeneous mixture of DNA o Can increase by factor of 10^9 within a few hours Procedure 1. Repeated cycles of template denaturation 2. Priming 3. Chain elongation Advantages: o Sensitive o Fast Limitations: o Only relatively short seq can be amplified o Must have some prior knowledge of DNA req in region Detect o Deletions Ducheness Muscular Dystrophy X-linked muscular degeneration disorder Dystrophin gene largest gene in human genome (24 mb)) Image

Microarray

PCR

 Detect SNPs

RNAi

DMD usually result of large deletions in dystrophin gene Deleted exons will fail to amplify in PCR o Detect DNA length expansion Repeat amplifications can be seen Products of PCR will migrate slower during gel electrophoresis Mutiplex PCR o Multiple primers in one sample SNP is a DNA seq diff b/w individuals Using PCR o Design allele specific PCR primers 3 most nucleotide determines specificity Result in products of diff amplicon size o Requires prior knowledge of mutation Using DNA seq Clinical sig of SNPs o Linkage to inherited disease and multifactorial traits (predisposition) Point to risk factors that may suggest preventive measures o Association with specific pathogen strains Hep C, HIV May signal resistance to specific therapies o Pharmacogenetics P450 polymorphisms Tamoxifen metabolism Variation in therapeutic efficacy could be explained by variation in drug metabolism Polymorphism analysis could guide dosing or selection Most eukaryotic cells have ability to use dsRNA to attack and degrade mRNAs o Using base-pair complementarity Utilize same pathway tt miRNAs follow: 1. dsRNA cut into smaller pieces by Dicer siRNA (23-25bp) 2. RISC complex picks up siRNA 3. siRNA unwound and one strand discarded 4. RISC complex containing antisense strand capable of annealing to mRNA with complementary seq 5. RISC attacks mRNA and fragments it 6. Results in mRNA inactivation NOTE: RISC-siRNA complex not consumed by mechanism o Very effective at gene silencing Cholesterol-targeted RNAi against apolipoprotein B o Tether siRNA to cholesterol o siRNA can get across cell memb o Picked up by RISC and gene expression shut down o Currently still done on mouse models