Chapter 10 using SIRI
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| 486345599 | DNA sequencing is used for | Detecting mutations, typing Microorganisms, identifying Human haplotypes and designating polymorphisms | |
| 486345600 | Targeted therapies will be directed at | Abnormal DNA sequences | |
| 486352352 | DNA sequencing define | The order of nucleotides in the DNA molecule | |
| 486352353 | Sanger method Discovered | Deoxy chain termination sequencing method | |
| 486363731 | Replication factor (RF) In regard to Dideoxy sequencing | Bacterial virus replicates by infecting E. coli, viral single-stranded circular | |
| 486363732 | Internal labeling Define | Fluorescent dye labeled nucleotides, added to the nucleotide sequencing reaction next. | |
| 486363733 | ddNTP Dideoxynucleotides | Is used for sequencing ddNTP lack the hydroxyl group found on 3' ribose C of the deoxy nucleotides (dNTP) | |
| 486370754 | Chain termination defined | DNA synthesis stop upon incorporation of a ddNTP Into the growing DNA chain | |
| 486370755 | Why does chain termination occur? | ddNTP Does not have a hydroxyl group at the 3' sugar carbon, so the 5'-3' phosphodiester Bond cannot occur & establish subsequent nucleotide (Create chain) | |
| 486374165 | Sections of DNA Created from PCR are often used as sequencing templates QA | PCR needs to be free of residual components such as primers and nucleotides | |
| 486376108 | QA:: PCR Amplicon's (Replicated DNA Sections) can be cleaned by ___, ___, ___. | Alcohol precipitation, enzymatic digestion with alkaline phosphatase, or if urines and washing on solid phase matrix (Bead or column) | |
| 486380311 | QA Running PCR Amplicon's on and agarose gel confirms the product being sequence and ____ | Cleans the template | |
| 486380312 | Components required for DNA synthesis are | Template, primer, enzyme, buffers, dNTPS. All four ingredients go in four separate tubes each two has individual ddATP, ddCTP, ddGTP, ddTTP | |
| 486407793 | ddNTP Concentrations are critical for generation of a readable sequence because | 1. high levels= polymerization will terminate too frequently Early along the template 2. Low levels= Infrequent or no termination will occur | |
| 486416707 | Stop buffer consists of | 20 mM EDTA To Chile cations and stop enzyme activity, formamide to denature the products of the synthesis reaction, and gel loading dies (bromiphenol blue and or xylene cyanol) | |
| 486416708 | QA Maintaining consistent reaction times will provide | Consistent band intensity | |
| 486416709 | Synthesize fragments are loaded onto a denaturing poly Accra Madlib gel, in ____ lanes | four adjacent lanes | |
| 486421275 | Sequencing latter define | The four-lane gel electrophoresis pattern of the products of the four sequencing reactions | |
| 486421276 | The sequencing ladder Deduces DNA sequences, showing smallest and largest Fragments in which location | Smallest fragments, fastest migrating, terminate closest to the primer, bottom of the gel Larger fragments found near 3', Near well | |
| 486424937 | Sequencing latter (polyacrymalide gel plate) is read from | Bottom to top, Smallest to largest Fragments of DNA, 5-3', Towards Wells where dNTP'S (A,T,C,G) were originally placed | |
| 486427633 | Cycle sequencing define | Use of thermal cyclers for the sequencing reaction using heat-stable enzymes such as Therminator and Thermosequenase | |
| 486650718 | Automated Fluorescent sequencing vs manual sequencing | Same chemistry, Using double-stranded templates and cycle sequencing. Automated cycle sequencing does Not require additional regions to start and stop the reaction therefore is easily adaptable to high throughput applications and automation | |
| 486650719 | QA: electrophoresis and reading of sequential ladder can be automated, ____ is required for reading the DNA sequence ladder. | Fluorescent dyes (instead of radioactive nucleotides to label the primers or sequencing fragments) | |
| 486660818 | Automated fluorescent sequencing, Using capillary or single Gelles Lane Is spread by | Each nucleotide (Reaction mix) Is read by It's own COLOR (A, T, C, G have a Different color each) | |
| 486660819 | Automated sequencing: dye primer vs dye terminator | Dye primer -5' END- uses labeled primers, the products of all four reactions are resolved together in one lane of gel or any capillary. Dye terminators - 3' END- One reaction to this necessary since they will be distinguished directly by the ddNTP on their 3' end | |
| 486666265 | After sequencing reaction using fluorescent dye terminators, excess dye terminators must be removed from the sequencing ladder or cleaned by ____ or _____ or _____. | Columns or beads or ethanol precipitation | |
| 486666266 | Sequencing ladder preparation protocol | Addition of my terminators to single file, sequencing cleaned, ladder completely denatured, | |
| 486670149 | Fluorescent detection equipment yields results as an _____. Software assigns one of four arbitrary colors | Electropherogram (Rather than a gel pattern) Passed by laserbeam, Beam excites fragment, dye emits it's florescents and is captured by the detector. | |
| 486674004 | Quality of electropherogram depends on what three things | 1 Quality of template, 2 Efficiency the sequencing reaction, 3The cleanliness of the sequencing latter | |
| 486674005 | Dye blobs ( bright flashes of fluorescence) - Are caused by? | Tell your to clean the sequencing ladder properly, They obliterate parts of the sequencing read | |
| 486676814 | QA Sequencing Both strands of DNA is important to | Confirm sequence data. This is critical for confirmation mutations or polymorphisms In a sequence | |
| 486676815 | Heterozygous mutations appear as | Two peaks of different color directly on top one another | |
| 486676816 | Heterozygous deletions or insertions appear as | (ex. BRCA Frameshift mutations) affect All positions of the sequence downstream of the mutation and are more easily detected | |
| 486676817 | Somatic mutations | I'm more difficult to detect they may be debited by normal sequences that mask the somatic changes | |
| 486682469 | Pyrosequencing DNA sequencing Does not have to make a | Sequencing ladder | |
| 486682470 | Bisulfate DNA sequencing or Methylation specific sequencing | Methylation is detected by comparing the treated sequence with an untreated sequence and noting where the sequence C/T base pairs are not changed to U/G |
