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