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215 MEIOSIS Recombination between Homologous Chromosomes 1. Occurs during gamete for...

215 MEIOSIS Recombination between Homologous Chromosomes 1. Occurs during gamete formation A a Alleles: A and a 2. Diploid progenitor cell generates four Bandb h aploid gametes b Candc 3. Meiotic recombination is frequent Red and Blue are homologous Chromosomes , one from each parent a A Interphou Anaphase I b B ~ ~ Early PN>pl--I ~ Telopl--I C Molecular Techniques @--· @)@) BASIC STEPS IN ISOLATING DNA FROM CLINICAL SPECIMENS 0 -pi--r Separate WBCs from RBCs, if necessary t lyse WBCs or other nucleated cells t Denature / Digest proteins t Separate contaminants (e.g., proteins, heme) from D NA t Precipitate DNA, if necessary t Resuspend DNA in final buffer DNA ISOLATION METHODS 1. Liquid phase organic extraction (phenol/chloroform) 2. Liquid phase non-organic extraction CHROMOSOMAL CROSSOVER 3. Solid phase procedures (GENETIC RECOMBINATION) ISOLATION AND PURIFICATION OF MJCLEIC ACID 1. Two chromosomes (paired up during 1. Sample source: prophase I of meiosis) exchange some a. Any tissue with nucleus portion of their DNA b. Blood collected in anticoagulant 2. Matching r egions of matching ❖ EDTA preferred chromosomes break then reconnect to ❖ DO NOT FREEZE WHOLE other chromosome (ex change of gen es) BLOOD 2. Storage conditions a. Store DNA in TE buffer at 4°C for weeks and at -20°C to - 70°C- for long term b. Store RNA in RNase free ultrapure water at - 70°C 216 NUCLEIC ACID ANALYSIS 1. DNA or RNA quantity , quality and REMEMBER! molecular size characterized b y Isolation of DNA from a. UV spectrophotometry b. Agarose gel electrophoresis Clinical Specimens c. Fluorometry Some = Separate d. Colormetric blotting L azy= Lyse NUCLEIC ACID QUANTITATION USING UV Dogs = Denature SPECTROPHOTOMETRY C an = Contaminants P lay = Precipitate 1. DNA and RNA absorb at 260 nm R ight = Resuspend 2. Proteins absorb at 280 nm 3. [DNA]= A2oo X dilution factor X 50 udm1 lOD unit at 260 run (A2oo)= 501tglml otDNA 4. [RNA]=A260 X dilution factor X 40 ug'ml lOD unit at 260 nm (.A260}= 40 uglml of RNA Concentration= ug of DNA or RNA per ml of hydrating solution Excrrple 1: DNA concentration A260 X Dilution Factor X 50 rig/ml DNA prepcrntion diluted l:200 yields A 200 reading of 0.200 DNA conc:entratim (ug/ml) = 0.200 d:>sai:xnce mits X 200 X SO ug/ml per rosort:x:nce mit = 200J ug/ml RNA concentration- A260 X Dilution Factor X 40 uglml Excrrple2 RNA prepcratim diluted 1:10 yields A2w reading of 0.500 Nucleic acid purity RNA Concentration = 0.500 ct>sorbcnce Lnit X IO X 40 ug/ml per rosort:x:nce mit = 200 ug/ml QUALITY FROM UV SPECTROPHOTOMETRY 1. A260/A230= mea sure of purity 2. 1.8-2.0 = good DNA or RNA RESTRICTION ENDONUCLEASES 3. Less than 1.8 = too much protein or 1. Bacterial enzymes cut or nick sp ecific other contaminants sites of a D A sequence ( "molecular scalpels'') CALCULATING NUCLEIC AOD YIELD 1. DNA/ RNA concentration multiplied by 2. R ecognize specific short sequences of DNA, usually 4 or 6 b ases but some are volume of hydrating solution 5, 8 or longer and cleave at or n ear Example: H DNA concentration from UV recognition site spectrophotometry = 250 uglml 3. R ecognition sequences are palindromes Volume of hydration solution = 0.1 ml Then DNA yield = 250 uglml X 0.1 ml= 25 ug  217 DNA MICROARRAYS (DNA CHIP) Cohesive Cohesive Blunt 1. Use hybridization technology to Ends Ends examine gene expression Ends (5' overhang) (3' overhang) 2. Arrangement of DNA sequences on BamHl Kpnl HaeIII solid support 3. E ach micro array contains thousands of t t t genes G Gjc C 4. Simultaneously monitor gene ~f..I.9.C GGTAC:C expression levels in all these genes C C~G G + CCTAG.'3 C;CATGG 5. Used for: l ♦ a. Gene expression studies b. Disease diagnosis c. Pharmacogenetics ( drug discovery) 6. Special instrumentation for a. Generation of micro arrays b. Analysis of results REMEMBER! Palindromes Palindromes are the same sequence on both DNA strands when read in either direction: 5' - GGTACC - 3' GENE CLONING (RECOMBINANT DNA TECHNOLOGY) 3'- CCATGG - 5' 1. I solating and amplifying a defined DNA sequence 2. Uses a vector, such as a plasmid and an appropriate host , such as E. coli DNA SEQUENCING 1. Determine order of nucleotides in a DNA molecule a. Maxam- Gilbert (chemical degradation) b. Sanger method (dideoxy chain termination) c. P yrosequencing (sequence b y synthesis) d. Next generation sequencing ( sequence single molecules of DNA in real time) ❖ Pacific Biosciences ❖ Oxford Nanopore ❖ Life Sciences Qdot technology