| Term | Definition |
|---|
| Ref Range | Values found in pts w/o disease |
| Analytical Sensitivity | "Detection limit" lowest measureable amount of nucleic acid |
| Analytical Specificity | detects a specific target |
| reportable range | range of values that will generate a postive result |
| Precision | same result repeated-reproducibility |
| Accuracy | correct results compared to external standard |
| Specimen Collection: addititve, testing: RED | add=none, test=chemistry, serum, viral antibodies |
| Specimen Collection: addititve, testing: Green | sodium heparin (freeze dried) test=immunology, virology |
| Specimen Collection: addititve, testing: Brown | add= sodium heparin, test= cytogenetic and molecular studies |
| Specimen Collection: addititve, testing: Lavender | add= Trip EDTA, test= virology, molec bio |
| Waived vs non waived | waived=simple methods, no risk/harm to pt if done incorrectly, not subject to routine inspection, Non waived (moderate and high complexity) <--what we do, must meet CLIA88 requirements, participate in proficiency tests, QC |
| preanalytical | test requirest, spec handling and collection, rejection of specimens, informed consent |
| analytical phase | procedure manuel, storage, contamination, lab design/practice, controls, test validation, manufactured |
| Post analytical phase | lab test reports, time of reporting, correction of errors, pt confidentiality |
| Stringency | Primary purpose to get rid of non specific binding in washing should be higher than during hybridization, Initial stringency wash: low stringency Removes non-specifically bound probe Second stringency wash: high stringency Removes partially bound probe |
| 2 ways of detection in Blotting | Radioactive, Chemiluminescense (Usually sandwich assay with enzymatic activity producing photons) |
| Stringency _____ with increasing temperature | increases |
| Stringency ____ decreasing salt concentration | increases |
| If you have a bone marrow sample that comes in Friday and nobody to process it until Monday what do you do with it | you can store it up to 5 days in 23 degrees celcius |
| If you have a blood sample that comes in and it can't be processed for 2 hours | sotre at 23 or 4 degrees celcius |
| Blood/bone/fluids under 2 hours | 23 or 4 degrees |
| Blood/bone/fluids under 5 days | 23 |
| blood bone fluids up to a week | 4 + denaturant |
| blood/bone/fluids 1-2 wks | -70 + denaturant |
| wbc 2-4 wks | -20 |
| Controls are | samples of known type or amount that are treated like and run with patient specimens. |
| With qualitative tests | positive, negative, and in some cases, a sensitivity control, are required. |
| In quantitative methods, | high-positive, low- positive, and negative controls are included. |
| For amplification procedures | amplification controls are required to avoid false-negative results. |
| Quantitative PCR methods that automatically analyze results require a | standard curve or dilution series of the positive control |
| In methods requiring detection of a target-specific product or relative amounts of target | internal controls are run simultaneously, preferably in the same reaction mix as the test specimen |
| Molecular quantitative methods should have a defined | dynamic range, sensitivity level, and accuracy |
| Assay levels that distinguish | positive from negative results (cut-off values) must also be well defined and verified at regular intervals. |
| The clinical sensitivity of an assay equals | TP /TP+FN X 100 |
| The clinical specificity of an assay equals: | TN /TN+FP X 100 |
| The clinical accuracy of an assay equals: | TN + TP / TN+TP+FN+FP × 100 |
| wbc up to 6 months | -70 |
| Tissue up to 2 hours | 4 |
| Tissue snap frozen up to 2 years | -70 |
| Tissue nitrogen vapor up to 2 years | -140--150 |
| Isolated RNA up to 30 days | -20 or -70 + DEPC treated water (not recommended) |
| Isolated RNA up to 6 months | -70 + ethanol |
| Steps in Organic DNA Isolation | 1) Lysis (NaOH, SDS) 2) Acidification (acetic acid, salt) 3) Extraction (phenol chlorform) 4) Precpitation (ethanol) |
| Steps in Inorganic Extraction | 1) Lysis (Tris EDTA, SDS) 2) Protein Precip (sodium acetate) 3) DNA precip (isopropanol) |
| What does phenol do in the extraction process | removes proteins/contaminates |
| what does chloroform do in the extraction process | denatures protein and removes residual phenol |
| what does isoamyl do in the extraction process | it is added to reduce chloroform foaming |
| What does SDS (Sodium dodecyl sulfrate) do during the extraction process | cell membrane break down by disrupting polar interactions |
| What does EDTA do in the extraction process | it weakens the cells by binding the divalent cations |
| What does NaCL do during the extraction process | it enables nucleic acids to precipitate out |
| What is the difference between Blot and Block transfer | Blot- transfer gel to membrane, Block-blocked all the spaces restricting non specific binding |
| 5 Primer Design Rules | 1) primers should be at least 15 base pairs long , 2) have at least 50% G/C content 3) anneal at a temperature in the range of 50-65 degrees C Usually higher annealing temperatures (Tm) are better , 4) forward and reverse primer should anneal at approximately the same temperature 5)Should not have repetitive regions |
| 3 steps and temps for PCR | Denature at 94, anneal at 55, extention at 72 |
| What happens if you run PCR too long | you reach a plateu when the reagents are depleted the products reanneal or the polymerase is damaged if you continue to run the sample too long it results in unwanted products accumulating |
| Primers determine the | specificity of the PCR reaction. |
| The distance between the primer binding sites will determine | the size of the PCR product. |
| Restriction Endonucleases:Eco RI- Isolated from? Recognition sequence? | E. coli, strain R 1st enzyme, G<cut> AATTC |
| Restriction Endonucleases: ECO RV-Isolated from? Recognition sequence? | E. coli, strain R 5th enzyme, G<cut> ATATC |
| Restriction Endonucleases: HIND III-Isolated from? Recognition sequence? | H. influenzae strain d, 3rd enzyme |
| What are the 3 restriction endonucleases | Eco RI, Eco RV, and HIND III |
| 3 Types of Restriction enzyme cutting | 5' overhang, blunt, 3' overhang |
| Restriction Enzyme Mapping The number of bands indicates | the number of restriction sites. |
| in Restriction Enzyme Mapping The size of the bands indicates | the distance between restriction sites |
| Methylation occurs as a result of | adding a methyl group (-CH3) to cytosine to form 5-methylcytosine |
| Methylation occurs almost exclusively with | cytosines located within CpG dinucleotides |
| Methylation is NORMALLY localized to | coding regions (CpG poor regions) while the promoter regions (CpG islands) remain unmethylated |
| Methylation of CpG islands results in | inhibition transcription of some genes (SILENCING) |
| Methylation of CpG nucleotide in promotor region can | turn off gene transcription -> silencing |
| How can methylation lead to disease | if a CpG promoter region becomes methylated it can block the tranSCRIPTion of a gene creating an absence of functional protein (the can also turn off tumor supprressor genes causing tumors) |
| Examples of Methylation Diseases | IFC, X-linked a-thalassemia/mental retardation , Rett Syndrom, Fragile X, Angelmann, Prader Willi, |
| 3 Blots for DNA Hybridization | Southern, Dot, and Colony or plaque transfer |
| Method for RNA hybridizatoin | Northern blot |
| method for protein transfer and hybridization | western blot |
| 5 Transfer methods | 1) Capillary 2) membranes 3) Plaque and colony 4) Vacuum 5) Electroblotting |
| Describe an ELISA pltae reader | protein and nucleic acid quantification or enzyme activity assays. A light source illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), and a light detector located on the other side of the well measures how much of the initial (100 %) light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest. |
| Describe Capillary Transfer | draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA. Only works with Charged nylon membrane...Not recommended when reprobing is desired |
| 2 different types of membrane which is preferred | Nitrocellulose; older not used much, Charged Nylon <--- what we prefer altho it has a higher background nylon binds more and is less fragile |
| Molecular Beacons | are hairpin-shaped oligonucleotides with a 3' dabcyl quencher and a 5' reporter. When the probe finds its target, the hairpin loop opens and generates a fluorescent signal. The overall signal is proportional to the amount of target. Molecular Beacons can be used for quantitative PCR and to distinguish single nucleotide sequences for SNP and allele analysis. |
| FRET Probes | The first oligonucleotide is labeled at the 3'-end (usually with fluorescein) and the second oligonucleotide is labeled at the 5'-end with a FRET acceptor .The first oligonucleotide hybridizes to the target in such a way that its 3'-end is separated from the 5'-end of the second oligonucleotide by1 base. If the target is present the labeled probes will hybridize with the target and FRET can occur. The fluorescence of the acceptor is mediated by the quencher that emits fluorescence at a longer wavelength than that of the acceptor. The fluorescence intensity is proportional to the amount of PCR product formed during the early exponential phase of PCR (threshold value). |
| Scorpion Primers | In the hairpin loop structure, the quencher forms a nonfluorescent complex with the fluorophore. Upon extension of the amplicon, the Scorpion probe hybridizes to the newly formed complementary sequence, separating the fluorophore from the quencher and restoring the fluorescence |
| Comparative Genomic Hybridization (CGH) detects | amplification (gains) or deletion (losses) ONLY |
| Southern Blot: Target, Probe? | DNA. probe=nucleic acid |
| Northern blot: Target, Probe? | RNA, probe= nucleic acid |
| western blot: target, probe? | protein, protein |
| Southwestern Blot: Target, probe | protein, DNA |
| Stringency describes | the conditions under which hybridization takes place. |
| Formamide concentration | increases stringency. |
| Low salt | increases stringency |
| Heat | increases stringency. |
| How do you determine the melting point (Tm) | 2(A+T) + 4 (C+G) |
| Ligase Chain Reaction | Isothermal, Probe amplification, Probes bind immediately adjacent to one another on template.The bound probes are ligated and become templates for the binding of more probes. |
| Branched DNA (bDNA) | Isothermal, Signal amplification, A series of hybridizations attaches multiple signals to each target molecule. |
| Hybrid Capture | Isothermal, Signal amplification, Immobilized DNA probes bind to RNA targets. The RNA:DNA hybrids are bound by labeled monoclonal antibodies. |
| Transcription-mediated Amplification (TMA) | Isothermal, Target amplification as RNA, cDNA is made from RNA target adding RNA polymerase promoter. RNA is synthesized from the cDNA template and can serve as a source of new cDNA. |
| Real Time PCR advantage over PCR is | quantifying the target. |
| RNA precautions | RNase activity can be found on all labwear liquid, seat, fingerprints and spit. Autoclaving and use of UV light will not sufficienty inhibit RNase. To prevent degradation use dedicated reagents/equipment. Wear clean gloves and change often. Buy RNase free reagents/equipment or treat glasswear/reagents/equipment to remove RNase (DEPC) |
| RNA should be stored at either | –20oC or –80oC in water or TE buffer |
| 230 nm | Detection of protein based upon the peptide bonds |
| 260 nm | Detection of nucleic acids (both DNA and RNA) |
| 270 nm | Detection of phenol contamination |
| 280 nm | Detection of protein, based upon the presence of aromatic amino acids |
| 320 nm | Background correction (particulates in solution or dirty cuvettes) |
| Purity | A260/A280 Ratio of 1.8-2.0 is pure |
| concentration of DNA = | A260 x .05 x dilution factor |
| Total DNA = | concentration of DNA x total volume of sample |
| Methylation Specific PCR (MSP) | Uses primers designed to be specific for methylated bisulfite-modified DNA These primers will not amplify unmethylated DNA Based on principle that oligonucleotides with mismatch at 3'-residue will not function as primers |
| Capillary Transfer | Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.DNA is now available for hybridization with labeled ssDNA or RNA probes. Possible only with positively charged nylon membrane Not recommended when reprobing is desired |
| 2 types of membranes | 1) Nitrocellulose; older not used much or 2) Charged Nylon: various types even tho it may have a higher background scientists feel nylon is better since it binds more and is less fragile. |
| Plaque and Colony Transfer | Membrane applied to the surface of a plate containing bacteria colonies or bacteriophage plaques. Colony transfer allows colonies to be grown on the membrane. These colonies are them lysed to liberate DNA bound to the membrane and are transferred to a second membrane where the DNA is denatured. |
| Vacuum blotting | Used for Southern blots, Most often used for dot/slot blots, Patient DNA or probe DNA then hybridized with either probe or patient DNA, |
| Electroblotting | Electric current applied through gel transfers DNA to membrane Preferred method for polyacrylamide gels |
| Gel electrophoresis is a | widely used technique for the analysis of nucleic acids and proteins. |
| Gel electrophoresis is a procedure that | separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. |
| In electrophoresis Nucleic acid moves from | a negative to a postive pole (anode) |
| DNA holds what charge | negative |
| Determination of gel migration | strength of the electrical field, buffer, density of agarose gel and the size of DNA |
| Agarose | -Derived from seaweed extractUsed for analysing DNA fragments between 100-20,000bp and by pulse field (10Mb) |
| Acrylamide | Produced by mixing acrylamide and Bis-acrylamide solution in the presence of TEMED catalyst. Most often used to separate very small molecules of 200bp or less. |
| concentration of gel/buffer will affect the ____ of fragments of different size ranges. | resolution |
| Loading Dye does what 3 things | 1) Terminates enzymatic reactions using EDTA, 2) Provides density for loading the sample to the well using glucose/sucrose 3)Monitoring the progress of electrophoresis using Bromethial Blue |
| Most frequently used buffer for gel electrophoresis | TAE buffer |
| Staining the Gel | Ethidium bromide binds to double stranded DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. |
| Polyacrylamide Gel Electrophoresis (PAGE) | Acrylamide, in combination with a cross linker, methylene bis-acrylamide. Finer resolution for smaller DNA molecules. |
| Gel Retardation (Gel shift or electrophretic mobility shift assay(EMSA)) | Used to investigate Protein-DNA interaction. Protein binds to small DNA sequences (10-100bp) and retards progress thro' gel. Specific binding proteins can be identified from cell homogenates using specific anti-bodies leading to "Supershifts". |
| Pulse Field Gel Electrophoresis (PFGE) | Normal electrophoresis can only separate DNA fragments up to 20kb. -Gels used are always agarose gels -PFGE can separate bands several Megabases long (1Mb= million base pairs) including whole chromosomes. |
| Capillary Electrophoresis (CE) | Separates solutes by charge/mass ratio.Capillary gel electrophoresis is used to separate nucleic acids. fused silica. More rapid, automated than slab gels. Electrokinetic injection of sample |
| Electrophoresis is used to | separate molecules by size and/or charge. |
| Nucleic acid fragments can be resolved on | agarose of polyacrylamide gels. |
| PFGE is used to resolve | very large DNA fragments |
| CGE is more | rapid and automated than slab gel electrophoresis. |
| The choice of electrophoresis method depends on | the type and size of sample. |
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