Term
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Definition
| release of nucleic acid from the cell |
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Term
| How do you release DNA (RNA)? |
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Definition
| breaking the cell and nuclear membrane = cell lysis |
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Term
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Definition
| removal of contaminating proteins, lipids, carbohydrates, and cellular debris |
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Term
| What are some organic isolation methods? |
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Definition
| high salt, low pH, and organic mixture of phenol and chloroform to dissolve hydrophobic contaminants (lipids and lipoproteins and DNA associated proteins) |
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Term
| What are the inorganic isolation methods? |
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Definition
| uses high salt and low pH to selectively precipitate proteins leaving DNA in solution |
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Term
| What are solid-phase isolation? |
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Definition
| uses a solid matrix to bind and wash the DNA called a spin column, which fits inside centrifuge tubes |
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Term
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Definition
| simple lysis for when high-quality DNA prep is prohibited |
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Term
| Who conceived the concept of amplification? |
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Definition
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Term
| What is the concept of PCR? |
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Definition
| double a test target by repetition to get 2^n |
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Term
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Definition
| DNA replication in vitro, one copy of double stranded DNA becoming two copies |
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Term
| What are the three steps of PCR? |
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Definition
| denaturation, annealing, and extension |
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Term
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Definition
| 90-96C, dsDNA separates into two single strands |
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Term
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Definition
| 50-70C, primer anneals to complementary sequences on the sample DNA |
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Term
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Definition
| 68-75C the polymerase adds dNTPs to the hybridized primer and replicated the template DNA |
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Term
| How many copies has the DNA been replicated into at the end of 1 cycle (3 steps)? |
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Definition
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Term
| What are the 5 components of PCR? |
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Definition
| Template, Primers, Nucleotides, polymerase, and buffer components (with Mg2+) |
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Term
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Definition
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Term
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Definition
| primes synthesis of the template and determines specificity, forward primer binds to target DNA, 3' is critical for extension |
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Term
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Definition
| thermostable enzyme that adds dNTPs to growing strand |
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Term
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Definition
| salts to provide optimal conditions for accurate replication and MgCl2 for primer annealing and enzyme activity |
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Term
| What are thermal cyclers? |
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Definition
| automated system designed to hold and rapidly change through incubation temperatures, 96 well plate format holds 0.2mL to 0.5mL tubes with heated lid to prevent condensation of the sample on tops of the tubes (add wax if no heated lid) |
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Term
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Definition
| more than one primer, multiple amplifications simultaneously |
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Term
| Reverse Transcriptase PCR |
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Definition
| starting material is RNA first has to be converted to cDNA, RT enzyme copies the RNA into RNA:DNA hybrid stand, used hairpin formation on the DNA strand to prime synthesis replacing the original RNA |
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Term
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Definition
| specimens limited in quantity and quality, two pairs of primers amplify a single target |
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Term
| Real-Time (quantitative) PCR |
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Definition
| monitor accumulation of PCR products in real time through fluorescence |
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Term
| What charge does DNA have in electrophoresis and why? |
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Definition
| each phosphate group on a DNA polymer is ionized making DNA a negatively charge molecule |
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Term
| Which way does DNA migrate in electrophoresis? |
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Definition
| toward the positive pole (anode) |
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Term
| What does the migration of DNA through agarose gel depend on? |
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Definition
| size of the DNA and spaces in the gel |
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Term
| what does buffer do in the agarose gel box? |
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Definition
| carries the current during electrophoresis, protects the gel and DNA molecules from damage by preventing severe pH fluctuations |
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Term
| What is the purpose of tracking dye? |
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Definition
| monitor the progress of the run, runs ahead of the DNA fragments, electrophoresis is terminated when dye approaches the end |
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Term
| Examples of density gradients |
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Definition
| ficoll, sucrose, and glycerol |
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Term
| What is the purpose of the density gradient? |
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Definition
| increase the density of the samples so its sinks into the well |
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Term
| What is bromophenol blue? |
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Definition
| tracking dye and density gradient |
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Term
| What is usually in lane 1? |
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Definition
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Term
| What is usually in lane 2? |
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Definition
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Term
| What is usually in lane 3? |
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Definition
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Term
| What does voltage depend on? |
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Definition
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Term
| What is used to visualize bands? |
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Definition
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Term
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Definition
| variable number of tandem repeats |
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Term
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Definition
| 15-70 bp sequences repeated 5-100x |
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Term
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Definition
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Term
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Definition
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Term
| What chromosome is D1S80 on? |
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Definition
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Term
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Definition
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Term
| Homozygous individuals band pattern? |
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Definition
| single band because equal repeat numbers are present on both homologues of chromosome 1 |
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Term
| Heterozygous individuals band pattern? |
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Definition
| 2 distinct PCR products, differing repeat numbers are present on the homologues of chromosome 1 |
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Term
| What is important to remember about EtBr? |
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Definition
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Term
| What are VNTRs and STRs used for? |
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Definition
| in forensic science, they are used for criminal investigations or paternity testing. it is a way to do a DNA "fingerprint" |
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Term
| What is proteinase K used for? |
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Definition
| The proteinase K will digest proteins in the sample, lysing the cells and inactivation other enzymes. This allows for isolation of DNA |
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Term
| Chelex is used to bind what kind of ions which can inhibit PCR reactions? |
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Definition
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Term
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Definition
| Polymerase chain reaction |
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Term
| 2 reasons why the bands would not show up on the gel? |
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Definition
| (1) chelex was extracted with the sample before PCR, the specimen was not in the chelex solution or (2) DNA was not obtained, DNases degraded the DNA before isolation |
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Term
| Which PCR step in most crucial and why |
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Definition
| annealing is most important because it is critical for specificity, the primers dictate the part of the template that will be amplified |
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Term
| If PCR is run for 36 cycles, how many copies of DNA? |
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Definition
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Term
| What is the buffer for gel electrophoresis? |
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Definition
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Term
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Definition
| it intercalates between the nitrogen bases in DNA and is carcinogenic |
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Term
| In which direction does the gel run? |
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Definition
| from The negative black cathode to the positive red anode |
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Term
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Definition
| agarose and polyacrylamide |
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Term
| Where do you see VNTR invesitgations in the clinical lab? |
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Definition
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Term
| What is the method of choice for engraftment monitoring? |
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Definition
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Term
| How can donor cells be monitored? |
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Definition
| following individual donor polymorphisms in the recipient blood and bone marrow |
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Term
| How can small VNTRs and STRs be easily detected? |
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Definition
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Term
| What happens before the transplant in engraftment/chimerism DNA testing? |
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Definition
| several polymorphic loci in the donor and recipient cells must be screened to find at least 1 informative locus, where the donor alleles differ from the recipient alleles |
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Term
| What happens after the transplant in engraftment/chimerism DNA testing? |
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Definition
| engraftment analysis, which is performed at specified intervals, where the blood and bone marrow is tested to determine the presence of donor cells using the informative loci |
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Term
| What is the preferred method of testing for engraftment/chimerism DNA testing? |
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Definition
| PCR amplification of STRs, resolution by capillary electrophoresis, and fluorescent detection |
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Term
| How can donor and recipient DNA for allele screening prior to transplant be isolated? |
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Definition
| blood, hair, or buccal cells |
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Term
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Definition
| when Donor and Recipient do not match |
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Term
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Definition
| when donor and recipient match |
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Term
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Definition
| one lane matches, one does not |
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Term
| Why is VNTR analysis performed before the transplant? |
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Definition
| find informative loci ( non-matching) that differ in pattern between the donor and the recipient |
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Term
| Why is VNTR analysis performed after the transplant? |
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Definition
| band patterns can be used to distinguish between graft failure, mixed chimerism, or full chimerism |
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Term
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Definition
| utilized for HBV, HCV, and HIV testing for blood donations, all in one run |
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Term
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Definition
| quantitate HIV in a specimen |
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Term
| in PCR, when we isolate DNA,we can go striaght to PCR. when the product is RNA, we must first convert it into DNA. How is this accomplished? |
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Definition
| reverse transciptase and t(th) polymerase |
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Term
| what organism was taq polyermase isolated from |
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Definition
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Term
| real-time PCR systems are equipped with fluorescent detectors which allow us to not only detect if a specific sequence is present, but also to measure PCR product as the reaction proceeds |
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Definition
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