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28 Cards in this Set
- Front
- Back
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Techniques That Test DNA?
Why do them? |
Restriction Enzyme Digestion,
Southern Boltting PCR Sequencing They all use to find changes in chromosomal DNA sequence |
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Two pairs A-T and G-C occur in DNA hybridization because of?
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The size limitiations of the Double helix and the hydrogen bonding that occurs
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Hybridization using complementary base pairing? How it works, and which tests?
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Make probe or primer that as same sqnc as short portion on one of chains of target DNA. Will bind to denatured target DNA by compl. bp.
Southern Blotting, PCR, sequencin |
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DNA hybridization creates?
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Unique pattern of h-bond acceptors and donors on its major and minor groove surface.
ie. guanine bases display H bond acceptors for + AA |
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DNA Replication requires?
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Primer at start site and 3' OH to be added to the last base.
3'OH acts as nucleophile and attacks phosphates to form phosphodiester bonds to release pyrophosphate, replacement of high energy bond with lower energy bond makes it favorable |
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Restriction enzyme digestion works how? Used for?
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Enzyme bind DNA at specific sequence Cleave it and cuts DNA in a particular fashion creating "STICKY ENDS" which can rehybridize with eachother or other DNA cut with same enzyme
Enzyme analysis can be used to ID DNA Can be used to recombine DNA |
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Sticky Ends?
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Ends made by restriction enzymes that can rehybridize to each other or to any other piece of DNA cut from the same enzyme
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Rule of restriction enzymes?
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DNA sequence determines which restriction enzymes can bind and where
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Restriction enzyme digestion creates fragments how are they read?
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Gel electropherisis seperates DNA by size,
Put in gel with anode at top cation at bottom, bigger fragments stay near top, larger go to bottom |
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Southern Blotting?
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Used to ID specific region of DNA--need probe to find that place
Target DNA is IDed based on restriction fragment size plus hybridization to a probe. Can ID one gene in entire genome |
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Southern Blotting Steps
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1. Digest Genomic DNA with restr. enzy. seperate fragments on gel
2. Denature and put on membrane 3. Incubate with probe (DNA homologous to target labeled with dye) 4.If DNA probe and comple. DNA which is the Target |
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Polymerase Chain Reaction (PCR)
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Amplifies defined region of DNA by repeated cycles of DNA replication
Specificity because region to be amplified is determined by short primers |
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Steps of PCR
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1. Denature DNA
2. Primers find target region bind by C-BP. 3.Primers prime synth. of Comp. strands of DNA in 5' to 3' 4.In next roun each of the new DS DNAs serve as templats for further amplification 5. End result=exponential ampl. of starting DNA |
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PCR ABSOLUTELY NEEDS
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A primer!, which are designed to hybridize to DNA flanking target sequence, 2 primers are used so taht both strands f DNA are replicated.
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DNA sequencing
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Exact order of BP in DNA, definitive DNA ID, ID Single BAse mutations or polymorphyims
Use dideoxynucleotides ddNTPS to make defined length sequences |
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ddNTPS are used in what?? WHY?
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DNA sequencing
They are chain terminators they lack a 3' OH. So a mixture of ddNTPS and dNTPS are used to synthesis new . Fragment size thus depends on ddNTPS position. Shorter fragments have ddNTP incorporated near primer |
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Trisomy 21 screening
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Prenatal diagnosis.
Analyze proteins in maternal serum. 2nd levels requirs obtaining fetal tissue throu amniocenticis. Ratio of chrom 21 DNA to chrom 12 DNA analyzed. Normal 1:1 Trisomy 21=== 1.5:1 |
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mRNA analysis tests?
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Northern Blotting
Q-RT-PCR Microarray analysis |
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Northern Blotting involved?
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DNA-RNA binding or hybridization by BP. PROBE-made with defined DNA or RNA sequence for RNA target
Used to ID on target RNA in a complex mixture. |
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Quantitative Realt Time PCR
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All mRNAs in cell are converted to cDNA by reverse transcriptase, and then amplified. A flourescent dye is incorporarted into the DS DNA so it can be monitored real time
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Microarray Analysis? What is it? How is it done and why?
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Allows you to survey a large number of genes at one time.
An microarray is made on a chip with know DNA oligs 25 bases long, these oligs match mRNA sequences that are to be analyzed. Up to 40000 genes on one chip. |
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Mircoarray analysis steps
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1.Make array
2. Isolate mRNa from reference sample and experimental sample. 3. Convert mRNA to cDNA 4.Label Ref/exp samples Incubate both pops on same chip. 6. Analyze ratios of experimental flouresence vs control sample for each gene. CAN BE USED TO ID PPL W/ BREAST CANCER RECURRENCE |
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Commercial test?
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Derived from the microarray analysisq
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RNA interference RNAi? Process?
Therapeutic RNAi pathways? |
Small non coding RNAs determine target.
Unwinds DB ogli finds 'guide strand which is incorporated into effector complex the RISC complex. KNOWN AS siRNA or miRNA. Enzymes in RISC complex (effector complex) go to target sequence and inactivate them thanks to guide strand Deplete endogenous RNAs Current siRNA trial for tumor growth suppression |
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Proteins analysis test?
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Western Blotting
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Antibodies made and bind to?
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Made by B cells as part of immune response, 2 heavy, 2 light chains, variable regions combine to recognize specif antigen and bind it, constant region combine to bind effector proteins recruited to neutralize antigen
antibodies bind foreing proteins and tag them for processin by immune system |
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Western Blotting
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Used to detect different protein characterisics, level of experssion, structural changes, post translational mods.
Id proteins by size and interaction with antibody |
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Two antibodies are used in Western blots what are they?
Important Why?? |
Primary antibody-unique to protein
Secondary antibody- recognizes constant region of primary antibody and binds to it. Can make new therapeutic agents |
