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38 Cards in this Set
- Front
- Back
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Agarose Gel Electrophoresis:
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separates molecules based on size – typically 500-10,000bp; sieving process; used for DNA analysis
1. Prepare a DNA sample for analysis (restriction digest or PCR) 2. Prepare a gel (melt agarose, pour into gel bed, add comb, harden) 3. Load DNA in wells, including markers 4. Turn on current, DNA will migrate a. DNA is (-) charged b. Use (+) & (-) electrical current charge c. Smaller molecules move faster → further down gel 5. Turn off current, stain gel, visualize bands a. Stain w/ethidium bromide b. Visualize w/UV light-box |
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Polymerase Chain Reaction
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preparation of lots of DNA of defined sequence from a small amount – exponential production
1. Denaturing: melt dsDNA → ssDNA 2. Annealing: allow primers to bind to target DNA a. Primers = complementary to 3’ end of DNA strand 3. Extension/elongation: thermostable DNA pol synthesizes new complementary DNA strand a. Thermostable DNA pol (taq) = functional @ high temps 4. Repeat process (anneal, extend, denature) 5. Visualize by AGE |
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Restriction endonucleases (RN):
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enzymes that cut DNA & RNA at specific locations; used for recombinant DNA biotechnology (cut/paste DNA sequences)
1. Function to kill off viral invaders 2. Cut at palindromic sequences (ex. GAATTC) a. Sticky/overhang ends: RN cuts asymmetrically, resulting in areas that don’t have any base-pairing b. Blunt ends: RN cuts right in middle of recognized sequence 3. Why doesn’t it kill native DNA? a. Restriction methylases: modify DNA at specific sequences by adding a methyl group. Sequence not recognized b/c RNs cleave unmodified DNA |
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Construct cDNA library
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representation of all expressed genes in a tissue, cell type or organism (contains only exonic portions; no promotes/introns/junk); representation of transcribed, mature mRNAs; used for understanding what portion of the entire DNA instructions are needed to make a specific tissue
1. Purify mRNA from tissue or organism 2. Reverse transcription: mRNAs → cDNAs 3. ss cDNA → ds cDNA 4. Add linker tails: segments of DNA containing RN cut sites are ligated to ends of ds cDNA 5. EcoR1 Rx (cuts restriction site?), ligation, transformation, plating |
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Hybridization screening:
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look for DNA of gene using nucleic acid probe
1. Library is plated onto many plates 2. Replica is transferred to filter disk 3. Inoculate filter disk with nucleic acid probe 4. Wash away unbound probe 5. Visualize probe → go back to original plate and select colony with plasmid of interest |
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Expression screening
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protein expression screened using antibodies; allows for identification of metabolic pathway components
1. Put library into expression vector (plasmid that contains promoter, origin of replication, and antibody resistance gene) 2. Insert is made into protein 3. Plate onto agar plates → transfer to filter disk 4. Incubate with labeled antibody that binds protein of interest → reactive colonies are detected |
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Chain termination
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analysis of cloned genes
1. Mix primer with DNA segment of interest 2. Add A, G, T plus equal amounts of C and *ddC (dideoxynucleotide) 3. Add DNA pol 4. Incubate → polymerization occurs 5. Chain termination occurs when there is *ddC a. *ddC lacks acceptor OH at 3’ end 6. Analyze by PAGE a. Run 4 lanes concurrently (one for each NT) b. Identifies NT positions in the template 7. (Today: automate system by using fluorescent labeled NT (one color/NT) → color of peak in graph identifies NT) |
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Southern Blot
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determine expression of DNA
- Used to detect CMT1A, fragile X syndrome, myotonic dystrophy, hereditary breast cancer 1. Prepare DNA (amplify with PCR) 2. Digest with RN 3. Analyze by AGE 4. Transfer to membrane (serves as replica to the gel) 5. Hybridize with labeled nucleic acid probe → wash off unbound probe 6. Detect position of bound probe using autoradiography a. Only band that is complementary to the probe lights up b. Darkness of band provides measure of amount of DNA in band |
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Southern Blot
**Used in RFLP analysis: capacity for a site to be cut w/RN; polymorphisms exist at RN restriction sites) |
- Used to detect fragile X syndrome
1. If differences in banding patterns b/t individuals → RFLP has been identified 2. Ex: Person 2 has a mutation such that EcoR1 will no longer cut, so they will have a longer fragment than Person 1 who doesn’t have the mutation 3. A person can be long (tye 1) or short (type 2) |
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Northern blot
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determine expression of mRNA
1. RNA is run on gel 2. RNA is then transferred to membrane 3. Hybridize with labeled DNA probe 4. Amount of DNA that binds is proportional to amount of mRNA in sample |
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Western blot
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characterizes proteins
1. Protein containing sample is analyzed by SDS-PAGE 2. Separated proteins are transferred to membranes 3. Incubate with labeled antibody → shows how much protein is in sample |
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Chip-based oligonucleotide hybridization (SNP analysis):
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1. Select 40-50 NT DNA segment for analysis
2. Make 33-43 tiled octamers (8NT in length) from that segment and spot each on chip 3. Label the 40-50NT DNA segment → hybridize with chip 4. Probe will only bind if perfectly complementary (8/8) a. If there is a SNP then there will be 8 blank spots on the chip (because the octamer will not bind) |
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Positional cloning:
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use linkage analysis to narrow down candidate region (RFLP, VNTR, SNP)→ obtain sequence in DNA in that region and identify all the genes→ prioritize the genes for mutation screening→ test candidate genes for mutation
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FISH:
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Observe chromosome translocation: hybridize fluorescent nucleic acid probe to chromosome
Observe overexpression of known gene: hybridize fluorescent nucleic acid probe to known gene → detect by fluorescent microscopy → extra dots indicate amplified gene (normally there would just be two dots) - Used to detect viral-induced cancer (HPV) or breast cancer (Neu2/Her gene) |
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IHC (Immunohistocytochemistry):
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uses antibodies to detect location of PROTEINS
Labeled antibody binds protein of interest in tissue → antibody-stained tissue is visualized by microscopy → darkness of reaction provides measure of protein abundance - Used to detect breast cancer and Gastrointestinal Stromal Tumor (GIST) |
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Chromosome painting
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identifies translocation event; paints each chromosome a different color so that a chimeric chromosome will have 2 colors
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Direct sequencing
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gold standard for detecting presence of disease-causing mutations
If 1 or few mutated NTs are the cause… 1. Amplify chromosome segment with PCR → directly sequence the region a. Exon amplified by PCR b. Mutation identified by automated sequencing If 3 mutation sites associated w/disease are on 3 different exons, then this requires 3 PCR reactions & 3 sequencing reactions… 1. Use Meta-PCR if need to stick together multiple exons |
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Heteroduplex-based analysis:
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Heteroduplex: structure formed when 2 DNA strands of different origin anneal to form ds segment
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dHLPC (denaturing high pressure liquid chromatography):
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analyze sample by size, rate of migration in sieving column; run under denaturation (perfectly matched samples will remain double-stranded)
- Used to detect muscular dystrophy 1. PCR amplify region of DNA to be analyzed 2. Denature & renature → form heteroduplex 3. Analyze by size on HPLC (run under denaturation) 4. Concurrent with sizing analysis, apply denaturation to separate imperfectly paired DNA a. Perfect matches = one main band (one peak) b. Heteroduplexes w/1 or more mismatched bases = extra peaks i. Mismatched base leads to lower melting pt |
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GE of heteroduplexes
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procedure like dHPLC but run under non-denaturing conditions
1. PCR 2. Heat denature/reanneal 3. Analyze by GE (under non-denaturing conditions) a. Normal result = one band on gel (perfectly matched) b. Heteroduplex result = 2 bands on gel |
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dGGE (denaturing gradient gel electrophoresis):
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electrophoresesced through a gel containing a gradient of chemical denaturant
- Used to detect muscular dystrophy, cystic fibrosis 1. PCR 2. Denature & renature → form heteroduplex 3. Analyze size on GE (gel contains gradient of chemical denaturant) a. Normal result = one band (perfectly matched) b. Heteroduplex result = band splitting (1 into 4) i. Extra bands represent melted strands |
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Enzymatic cleavage of heteroduplexes
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- Used to detect cystic fibrosis
1. PCR 2. Denature & renature → form heteroduplex 3. Mismatched bp can be cleaved by chemicals or nucleases 4. Heteroduplexes are denatured 5. Analyzed by sizing on a column or by GE a. Heteroduplex result = small blips on the graph which are indicative of shorter cleaved fragments |
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PTT:
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test for translated protein whose size is smaller than normal
- Used to detect Duchenne muscular dystrophy, hereditary breast cancer, familiar adenomatous polyposis 1. PCR 2. Incorporate bacteriophage promoter for in vitro gene expression 3. In vitro transcription/translation (label protein) 4. Protein analysis by SDS-PAGE (measures size) a. Truncated protein result = shorter than normal bands (indicate presence of premature stop codon) i. Shorter protein travels faster & further down than control lane (shows normal protein size) |
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Multiplex PCR:
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for genetic diseases resulting from large deletions in which one or more exons are lost; uses many primer pairs to examine multiple exons concurrently
- Used to detect muscular dystrophy Prepare many primers that will flank many regions of the chromsome→ PCR (anneal, extend, denature)→ analyze on GE→ stain with EtBr Deleted exons result: missing bands (primer cannot bind to deleted target)→ compare with control lane |
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SSCP (single strand conformation polymorphism):
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in this case, polymorphism means different conformations of ssDNA; when DNA is denatured into ss, DNA can fold → conformation can affect rate of migration through gel; used to detect disease causing mutations
- Used to detect cystic fibrosis 1. PCR 2. Denature DNA to get ssDNA 3. Analyze by GE a. Normal result: 2 bands on gel b. SNP result: >2 bands on gel (extra bands = NT differences = disease causing mutations) |
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Allele-specific oligonucelotide hybridization (ASO):
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can test for numerous disease causing mutations in a single analysis
- Used to detect cystic fibrosis, hereditary breast cancer, familiar adenomatous polyposis 1. Parallel to SNP analysis with oligonucleotide microarrays 2. Mutation is identified by non-hybridized spot on the filter a. Perfect match = hybridization occurs b. Single bp mismatch = no hybridization |
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Allele-specific PCR (ARMS; amplification refractory mutation system):
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to test for disease causing alleles
- Used to detect cystic fibrosis 1. Design PCR primers that will only bind disease causing allele → carry out PCR reaction 2. Analyze by GE a. Primers will bind and bands will show up on gel = if disease causing allele is present b. Absence of bands = person does NOT have disease-causing allele; they have the normal allele |
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Run-on Transcription assay:
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measures # of RNA pol bound to & transcribing a gene; measures which gene is made (darkened areas) & how active the promoter is (degree of darkness); snapshot of DNA gene showing its transcriptional activity measured by # of associated, active RNA pol
1. Isolate nuclei 2. Add radioactive label 3. Incubate (with target DNA) for one round of transcription 4. Labeled RNA to complementary DNA on filter a. Able to measure amount of RNA pol on the DNA which is direct measure of on-going transcription |
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Reporter gene expression assay
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measures capacity of PROMOTER
1. Fuse promoter upstream of reporter gene (commonly luciferase) 2. Transfect the construct into plasmid (vector) 3. Incubate 4. Measure gene expression by the amount of protein activity (light emission) **Able to determine where important elements are in promoter by running test with truncation mutations → progressively shop away at promoter and run expression assay and then compare the amount of light emitted (protein activity) - Changes in protein activity reveal at what point you have cut off critical sites in the promoter |
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Gel retardation assay
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in vitro; measures physical binding of TF to DNA promoter; based on fact that MW/size of short segment of DNA become larger when protein is bound → slower in gel
1. Synthesize DNA segment w/hypothetical TF binding site 2. Add TF 3. Incubate 4. Run on PAGE a. Protein-bound DNA will not migrate as far as free DNA |
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DNA footprinting:
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in vitro; measures binding of TF to DNA promoter; based on fact that TF protects DNA from nuclease/chemical cleavage
1. Radiolabel one end of DNA segment 2. Add TF 3. Incubate 4. Add chemical/nuclease to partially cleave DNA 5. Analyze w/PAGE & compare to naked DNA a. Where TF binds & protects DNA from cleavage, there will be a BLANK spot = footprint |
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ChIP assay (Chromatin Immunoprecipitation Assay):
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analyzes TF binding in LIVING cells
1. Chemically cross-link proteins to DNA in situ 2. Partially cleave DNA into segments that are 500-1000NT in length 3. Incubate with antibody that will bind to TFs that are bound to the DNA 4. Recover any anytibody-TF-DNA complexes by immunoprecipitation (centrifuge) 5. Undo cross-links to remove proteins from complex 6. Amplify region of DNA using PCR 7. Analyze by AGE or PAGE |
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Microarray analysis:
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gold standard for mRNA expression analysis; compares mRNA expression patterns b/t different cell types
1. Begin with matched pair of cell types (normal and cancerous) 2. Prepare sample of mRNA from each cell type 3. Convert mRNA to cDNA using RT 4. Fluorescent label for the two cell types a. Normal = green, cancerous = red 5. Prepare microarray: each circle contains millions of copies of ONE gene so in theory there should be ~30,000 circles 6. Mix the two cDNA pools and hybridize them together with the microarray circles → 4 results are possible: a. No gene is expressed → no color b. Normal gene is expressed → green dot c. Cancerous gene is expressed → red dot d. Both cell types are expressed → yellow dot (mix of red/green) |
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RT-PCR:
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characterizes abundance of specific mRNA
1. RT primer binds target mRNA 2. Complementary DNA strand is synthesized 3. Denature and remove mRNA 4. Add PCR primers to continue with process of cDNA strand (anneal, extend, denature) 5. Repeat → millions of DNA segments a. Amount of DNA produced is proportional to amount of mRNA in sample |
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Summary of Genetic Diseases & Tests
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Genetic diseases & tests:
1. Breast cancer – test for amplified her2/neu receptor (FISH, IHC, gene chips) 2. Gastrointestinal Stromal Tumor, GIST (IHC → gleevec) 3. Chromic Myeloid Leukemia, CML (chromosomal coloring → gleevec) 4. Cervical cancer – determine HPV levels (FISH) 5. Muscular Dystrophy (dHPLC or dGGE, multiplex PCR; for Duchenne MD: PTT) 6. Cystic Fibrosis (SSCP, heteroduplex analysis, dGGE, allele-specific PCR, cleavage of heteroduplexes) 7. Charot-Marie-Tooth Disease Type 1A, CMT1A (Southern Blot, FISH, pulsed-field GE) |
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Common trinulceotide repeat expansion (TRE) diseases:
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1. Fragile X Syndrome (methylation sensitive RFLP/Southern Blot)
2. Huntington’s Disease (PCR) 3. Myotonic Dystrophy (Southern Blot/RE) **Use PCR, Southern Blotting |
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Other genetic diseases commonly tested for using gene tests:
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1. Hereditary breast cancer (Southern Blot, PTT, ASO)
2. Familiar adenomatous polyposis (PTT, ASO) 3. Hereditary nonpolyposis colorectal cancer syndrome, HNPCC (MSI, sequencing) |
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Polyacrylamide GE (PAGE):
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separates molecules based on size, but more precise than AGE; process = same as AGE, but differences are:
1. Run using denaturing conditions (ssDNA) 2. Pores = much smaller, so only useful for molecules <1000bp 3. Used for DNA sequencing |
