DNA Microarrays (DNA Chip) PDF

217 DNA MICROARRAYS (DNA CHIP) Cohesive Cohesive Blunt 1. Use hybridization technology to Ends...

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 218 SINGLE NUCLEOTIDE POLYMORPHISMS (SNP) SIGNAL AMPLIFICATION TECHNIQUES 1. Mutation of a single nucleotide 1. Branched DNA (bDNA) (A,C,T,G) 2. H ybrid capture assay (HCA) 2. Some associated with various phenotypic THE POLYMERASE CHAIN REACTION (PCR) differences a. Propensity towards disease 1. Denaturation (95°G) - separation of b. Drug resistance target dsDNA through a. Alkaline pll 3. Over 5 million S P locations identified h. Ionic strength of high salt solution in human genome c. Elevated temperature 2. Primer annealing at 50-60°C - Minisatellite: Tandem rePNtt of seqi,tnets that -vary from 14 to 100 base pairs in length h ybridize the primers to the single-...ACGATATCGGACCAATCGATCGGACCAATCGATCGGACCAATCGTAGGT... stranded template +...ACGATATCGGACCAATCGATCGGACCAATCGTAGGT... 3. Extension (72°G) - polymerize the Polymotphism."YlrialMe number of rtpms primer into the full-length gene of Microsatellite: interest Shott sequences of tand m ropN!s , 19. CA,epem...TGCCATAGCACACACACACATTAGTTAG... ♦...TGCCATAGCACACAC.ACACACACATTAGTTAG... 4. Each cycle doubles amount of DNA Potymorphiim:variable number of CA repeab SNP: Single nucleotide pol~morphlsm...TGTACCAAGT... REMEMBER! ♦...TGTAACAAGT... Denature by the SEA Salt (high ionic strength) FLUORESCENT IN-SITU HYBRIDIZATION (FISH) Elevated temp 1. H ybridization of a fluorescent DNA probe to its complementary DNA in Alkaline pH Denature morphologically preserved tissue or ~ e:;,4 cells 2. Detect and localize presence or absence CONTROLS FOR PCR of specific DNA sequence or chromosome 1. Blank r eaction a. Controls for contamination 3. Ahle to examine metaphase chromosome b. Contains all r eagents except DNA spreads as well as interph ase (non- template dividing) cells 2. egative control r eaction a. Controls for specificity of the Amplification Techniques amplification reaction b. Contains all r eagents and a DNA COPY NUMBER AMPLIFICATION template lacking the target sequence 1. Polymerase chain r eaction (PCR) 3. Positive control reaction 2. Ligase chain reaction (LCR) a. Controls for sensitivity h. Contains all r eagents and a known 3. Nucleic acid sequence-based target-containing DNA template amplification (NASBA) REVERSE TRANSCRIPTION POLYMERASE CHAIN 4. Transcription-mediated amplification REACTION (RT-PCK) (TMA) 1. Synthesis of complementary piece of 5. Strand-displacement amplification DNA (cDNA) from RNA b y r ever se (SDA) transcription (R1) 2. Amplify the cDNA target sequence by PCR  219 Target DNA Reaction mixture contains target DNA sequence to be amplified, two primers 1i1 1 11 111 11111111111 I'I (P1, P2), and heat-stable I raq ·~ P1 ~ Taq polymerase Reaction mixture is heated I I 111 I I IIIl to 95·C lo denature target DNA. Subsequent cooling......... 111 I I to 37 C allows primers to hybridize to complementary sequences in target DNA I bi il 111 , l i ~ I ~ 1111 When heated to 72°C, Taq polymerase extends complementary ) strands from primers I ~ I ,.,,,.,. I 1111 111 j 1111 j ! I I First ~~nt~~s~~~;~~eo~esults ~ I target DNA sequence ( ~· Denature Il I I I 11 i I ' DNA Hybridize Q) primers u>, (.) Extend "C C: 11 ,. , 1 : I ii I I j I I new DNA 0 strands 0 qi t/l 11 I Second synthesis cycle results in four copies of ) I I !I target ONA sequence DNA Amplification Using Polymerase Chain Reaction Reprinted with permission Copyright Holder is Cold Spring Harbor Laboratory Press REAL-TIME PCR / QUALTITATIVE PCR (qPC/('J NUCLEIC ACID SEQUENCE BASED AMPLIFICATION 1. Qualitative and/or quantitative (NASBA) technique 1. A nucleic acid sequence based 2. Detects fluorescent reporter molecule amplification technique that amplifies after each cycle RNA and rRNA Strand Displacement Amplification (SDA) 2. Direct detection of RNA viruses/ H CV and HIV 1. Target amplification u ses heat denaturation, annealing and extension HYBRID CAPTURE 2. Creates an altered target with a 1. Nucleic acid amplification technique r estriction endonuclease r ecognition used for RNA or DNA applications site. 2. Solid phase using chemiluminescence 220 BRANCHED CHAIN DNA (bDNA) 1. Uses a series of hybrid probes to elicit a signal amplification- chemiluminescence REMEMBER! 2. Detect specific RNA sequences Tech PENS Load Gels! Electrophoresis Technology P = Pore size of gel ELECTROPHORESIS OF NUCLEIC ACIDS - E = electric field N = negative DNA charge 1. Separation based on size and charge through a sieve-like matrix (agarose 01· polyacryla1nide) S = size of DNA CJ CJ CJ CJ - 2. Migration in electrical field at a rate inversely proportional to loglO of molecular size (number ofbase pairs) 3. DNA (negatively charget{) migrates toward anode (positively charged) FACTORS AFFECTING MIGRATION RATE Tortoises are big and slow Bunnies are small and fast 1. Matrix type and porosity (%) of the gel (gel castinB) Movement through a gel depends on the size of the DNA particle, its charge, and the pore size of the 2. Net charge of nucleic acid molecule gel. All negatively charged DNA particles move toward the anode, but the larger pieces have a 3. DNA conformation harder time squeezing through the small pores in the gel and cannot move as fast, i.e., as far, as the 4. Electric field strength smaller pieces. 5. Temperature of gel Blotting Techniques 6. ucleic acid base composition SOUTHERN BLOT 1. Detects specific DNA sequences 7. Presence of intercalating dyes 2. DNA denatured in the gel by an 8. Type and strength of buffer increase in pH PULSED FIELD Ga ELECTROPHORESIS OF DNA (PFGE) 3. DNA transferred to a membrane by 1. Analysis of DNA fragments up to 100 capillary action with a high salt kb in size solution 2. Separation accomplished using a pulsed electrical field 4. Labeled complementary probe used for 3. PFGE commonly used for genotyping detection prokaryotes Paper towels Nitrocellulose I filter membta ne Gel Methods, instruments, reagents & controls Sponge Routine and special procedures to verify test results High Salt Solution