DNA Sequencing Chapter 10 Exam Questions and Answers Latest Update Graded A+

DNA Sequencing Chapter 10 Exam Questions and Answers Latest Update Graded A+ chemical (maxam-gilbert) sequencing - Answers ds or ssDNA; one end radioactively labeled; template aliquotted into four tubes and treated with four different chemicals and a strong reducing agent like piperidine, causing the DNA to break at specific nucleotides; fragments are then resolved on polyacrylamide gel and read 5' to 3' (bottom to top) cons of chemical (maxam-gilbert) sequencing - Answers low throughput, toxic chemicals dideoxy chain termination (sanger) sequencing - Answers modification of the DNA replication process in which a short synthetic ssDNA primer complementary to sequence 5' to the ROI is used for priming dideoxy sequencing reactions; modified ddNTP derivatives lacking the 3' OH are added to the rxn mixture (containing normal dNTPs) which causes chain termination; manual sanger takes place in four tubes (ACTG) containing only one type of nucleotide internal labeling - Answers incorporating 32P- or 35S-labeled deoxynucleotides in the nucleotide sequencing reaction mix Why is the ratio of ddNTPs to dNTPs crucial? - Answers Too many ddNTPs and polymerization will terminate too frequently early on in the template Noo low ddNTPs and little or no chain termination will occur What is used to stop the chain reactions? - Answers 20 mM EDTA to chelate ions and stop enzyme activity Formamide is used for... - Answers denaturing the products of the synthesis reaction How are the chain reactions in sanger sequencing read as a sequence? - Answers The sets of synthesized fragments are loaded onto a denaturing polyacrylamide gel; products of the four reactions are loaded into separate adjacent laneslabeled ACTorG corresponding to the ddNTP used; fragment patterns visualized by the signal on the 32P-labeled primer or nucletide; the four lane pattern of called a sequencing ladder and is read to deduce the DNA sequence In which direction is a Sanger polyacrylamide gel read? - Answers from the bottom of the gel (fastest migrating = smaller fragment) where the first ddNTP was terminated to the top of the gel where the largest fragments are How many bases per sequence can be read in manual sanger sequencing? - Answers 300-400 cycle sequencing - Answers A modified automated version of the Sanger method (12000 bases per min). The four deoxynucleotides are fluorescent labeled, polymerization in a single tube, resulting mixture separated using capillary electrophoresis in a single narrow tube gel, then read by laser beam. Color sequence is converted to DNA sequence by computer. dye primer sequencing - Answers four different fluorescent dyes are attached to four separate aliquots of the primer; dye molecules are are attached covalently to the 5' end of the primer during chemical synthesis resulting in four versions of the same primer with different dye labels; requires four separate rxn tubes and then subjected to cycle sequencing Dye Terminator Sequencing - Answers utilizes fluorescent labeling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labeled-primer method. Laser and detector "read" the fragments. Labeled at 3' end. A label - Answers green C label - Answers blue G label - Answers black or yellow T label - Answers red How are sequencing ladders cleaned of excess dye terminators? - Answers columns, beads, or ethanol precipitation Why are cleaned sequencing ladders resuspended in formamide and heated to 95C for 2-5 min? - Answers Denaturing to ensure the fragments are resolved according to size Advantages of Sanger sequencing using color coded dyes - Answers products of sequencing rxn can be loaded onto one capillary or gel lane; increases throughput and eliminates lane to lane migration variation True or false: fluorescent detection equipment yields an electropherogram rather than a gel pattern - Answers True; machine calls the bases from smallest to largest; fragments that first pass the detector; senses color dye blobs - Answers bright flashes of fluorescence that result from a failure to clean the sequencing ladder properly how does a heterozygous mutation appear on a Sanger electropherogram? - Answers two peaks of equal height but different color directly on top of each other; overlapping peaks should be about half the height of the surrounding peaks pyrosequencing - Answers Detects addition of a nucleotide to the end of a synthesized strand of DNA by production of light when the phosphodiester bond is formed between dNTP and primer, converting pyrophosphate to ATP which is them used to generate a luminescent signal pyrogram - Answers results of pyrosequencing that consists of single peaks of luminescence associated with the addition of the complementary nucleotide what is pyrosequencing most often used for? - Answers short to moderate sequence analysis; mutation or SNP detection and typing; infectious disease typing, HLA typing