DNA SEQUENCING AND MICROARRAY EXAM QUESTIONS AND ANSWERS GRADED A+

DNA SEQUENCING AND MICROARRAY EXAM QUESTIONS AND ANSWERS GRADED A+ DNA SQUENCING - Answers It determines the exact sequence of nucleotides/ bases in a DNA molecule ACTG (nucleotides) - Answers Encode biological informations sequence of DNA - Answers establishing the__________ is key to understanding the functions of genes and other parts of the genome -detection of mutations -typing microorganisms -identifying human haplotyles -designating polymorphisms - Answers Uses of DNA Sequencing Direct determination of nucleotide sequence or DNA sequencing - Answers Most effective molecular method to identify genetic lesions (mutations) -Maxam-Gilbert Method -Sanger Method - Answers Manual Sequencing Methods (used most extensively) Allan M. Maxam and Walter Gilbert (Late 1970s) - Answers Maxam-Gilbert Method developer dsDNA or ssDNA version of DNA region; with one end radioactive labeled (32p) - Answers MAXAM-GILBERT is a chemical sequencing that requires: MG Method template - Answers Aliquoted into four (4) tubes; each was treated w diff chemical with/without high salt -Dimethylsulphate -Formic Acid -Hydrazine -Hydrazine + salt - Answers Specific base reactions (MGS) base modifier 10% piperidine - Answers Strong reducing reagent that causes ssDNA to break at specific nucleotides upon addition Dimethylsulphate; GUANINE (chain) - Answers breaks the chain of _________ by methylating G at 25 degrees celsius for 4 minutes Formic Acid; Guanine + Adenine breaks - Answers breaks the chain of _______ by protonating purines at 25 degrees celsius for 5 minutes Hydrazine; Thymine + Cytosine - Answers breaks the chain of _______ by spliting pyrimidine rings at 25 degrees celsius for 8 minutes Hydrazine + salt; Cytosine - Answers breaks the chain of _______ by spliting only Cytosine rings at 25 degrees celsius for 8 minutes formamide gel loading (AFTER MABREAK) - Answers Maxam- Gilbert Method: After reactions, piperidine is evaporated; contents of each tube dried and resuspended in ______________ denaturating Polyacrylamide gel - Answers Maxam- Gilbert Method: fragments are then separated by size on a Denaturating conditions - Answers Prevent the ssDNA fr hydrogen bonding w one another or folding up so that they migrate through the gel strictly acc to their size Migration speed - Answers Maxam- Gilbert Method: it is important bc single-base resolution is req to interpret the sequence properly Gel dryer - Answers After electrophoresis; this is where sheet of filter paper was dried after gel was removed Light sensitive film - Answers where dried gel will be exposed Wet gels - Answers Maxam- Gilbert sequencing: can be Exposed directly Bottom to the top - Answers Maxam- Gilbert sequencing: The gel is read fr Maxam- Gilbert Method - Answers -not practical for high throughput sequencing of long fragments -relatively efficient way to determine short runs of sequence data Hazardous chemicals hydrazine and piperidine - Answers Require more elaborate precautions for use and storage Dideoxy chain termination sequencing method - Answers Maxam- Gilbert Method is replaced by: Sanger method - Answers THE ORIGINAL dideoxy chain termmination sequencing method required a single-stranded template Classical Sanger Method Modified Sanger Method - Answers 2 classification of Sanger Method M13 BACTERIOPHAGE - Answers Templated up to a few thousand bases long could be produced using (a bacterial virus with ssDNA genome) Replication factor - Answers The virus replicated by infecting E. coli, in which the viral ss circular