Biochemistry 2280A Lecture Notes - Lecture 27: Chain Termination, Dna Profiling, Stop Codon

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raspberrywhale574

10 Dec 2016

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To be sure that yfg is correct, it should be sequenced. Two methods were developed in the early 70s to sequence dna: chemical sequencing (maxam and gilbert, dideoxy or chain termination sequencing (sanger) method of choice. Involves the enzymatic synthesis of a dna strand in the presence of base specific chain terminators. Dideoxy ntps (ddntps) act as chain terminators. Dideoxy lacks 3" oh, prevents strand extension at 3" end. Oligonucleotide primer that anneals to the template. When you add a ddatp chain termination occurs. Billions of reactions in tube, goes on over and over. At each base where a da should be inserted into the growing chain, there is a. 1/500 chance that a dda will be inserted and the chain terminated. A collection of dna molecules is made that all have the same 5" end but differ at the a position at which they stop. Do 4 separate reactions, each with a different ddntp.

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3. What allows chain termination to occur at each base of thetemplate during an automated sequencing reaction?

a. the presence of a relatively low concentration of ddNTPs

b. the presence of a relatively low concentration of dNTPs

c. the presence of a relatively high concentration of ddNTPs

d. the presence of a relatively high concentration of dNTPs

4. In automated fluorescent sequencing, what would happen if thefluorescently labeled ddATP was inadvertently omitted from thereaction?

a. DNA polymerase does not function without equal concentrationsof the four nucleotides

b there would be gaps in the sequence where thymines werepresent in the template DNA strand

c. the sequence would end where the first thymine wasencountered on the template DNA strand

d. the sequence would end where the first adenine should beincorporated

bluehound936

31. Why does incorporation of a dideoxynucleotide into a growingDNA polymer terminate strand synthesis?

a. There is no longer a free 5âphosphate to which to add the next nucleotide.

b. There is no longer a free 3â hydroxyl to which to add thenext nucleotide.

c. There is no longer a free 3â phosphate to which to add thenext nucleotide.

d. There is no longer a free 5â hydroxyl to which to add thenext nucleotide.

2. The output from an automatedsequencing reaction shows a series of fluorescent peaks. Whenconsidering the data output, why are some peaks larger thanothers?

a. Chain termination occurred more frequently at the basecorresponding to a large peak, producing a relatively largerfluorescent signal.

b. The primer had multiple bases fluorescently labeled forproducts corresponding to the larger peaks.

c. More bases were fluorescently labeled in the DNA strandscorresponding to the larger peaks.

d. Some fluorescent colors are brighter than others.

3. What allows chain terminationto occur at each base of the template during an automatedsequencing reaction?

a. the presence of a relatively low concentration of ddNTPs

b. the presence of a relatively low concentration of dNTPs

c. the presence of a relatively high concentration of ddNTPs

d. the presence of a relatively high concentration of dNTPs

4. In automated fluorescent sequencing, what would happen if thefluorescently labeled ddATP was inadvertently omitted from thereaction?

a. DNA polymerase does not function without equal concentrationsof the four nucleotides

b there would be gaps in the sequence where thymines werepresent in the template DNA strand

c. the sequence would end where the first thymine wasencountered on the template DNA strand

d. the sequence would end where the first adenine should beincorporated

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Biochemistry 2280A Lecture Notes - Lecture 27: Chain Termination, Dna Profiling, Stop Codon

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