BIOLOGY 172 Lecture Notes - Lecture 21: Shotgun Sequencing, Sanger Sequencing, Dideoxynucleotide

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taupecod750

20 Dec 2018

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Used to determine to determine the sequence of nucleotides in a stretch of dna. Need: template dna, primer, dna polymerase, deoxynucleotides, dideoxynucleotides. Dideoxynucleotide - lacks 3" hydroxyl, cannot be enlarged by dna polymerase. Nucleotides cannot be added to the 3" end. Dideoxynucleotides are died with different fluorescent dyes. Need to know the beginning sequence (to create the primer) Two options when adding to the primer. Dctp can be added - chain can be elongated. Ddctp can be added - chain terminated. Creates dna chains with a range of sizes, each labeled with a fluorescent dye. Run sequences through a gel and a machine orders the lengths & reads the colors. From this, you can deduce the sequence. Single sequence reaction can yield a few hundred to thousand base pairs. Human genome contains 3 billion base pairs. Shotgun sequencing - cut genome into small fragments, sequence them, assemble the entire sequence.

Related Questions

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

azuresnake426

1. Which of the following is the correct sequence of events (from start to finish) in cellular fatty acid oxidation?

A. Fatty acid activationÃ carnitine shuttleÃ beta-oxidation

B. Beta-oxidationÃ carnitine shuttleÃ Fatty acid activation

C. Fatty acid activationÃ carnitine shuttleÃ beta-oxidation

D. Carnitine shuttleÃ Fatty acid activationÃ beta-oxidation

2. What reaction is catalyzed by lipoprotein lipase, or lipase?

A. Hydrolysis of TAGs into fatty acids and glycerol

B. Hydrolysis of TAGs into amino acids and glycerol

C. Hydrolysis of cholesterol in to fatty acid and water

D. Hydrolysis of cholesterol into acetyl-CoA

3. How do fatty acids of different carbon lengths cross into the mitochondrial matrix for oxidation?

A. Small FAs of 10 carbons or fewer can freely diffuse through the membrane

B. Large FAs are cleaved into two smaller units that are actively transported across the membrane

C. A membrane-bound transferase catalyzes the conversion of long-chain FAs and FA-carnitine esters, which are transported across the membrane via a specific carrier

D. The breakdown of large FAs starts outside the matrix, allowing for the smaller particles to pass easily through the membrane

4. How can any RNA nucleotide be easily distinguished from a DNA nucleotide?

A. RNA nucleotides have a unique phosphodiester bond linkage

B. RNA nucleotides have uracil nitrogenous bases

C. RNA nucleotides have an -OH on the 2â carbon of the ribose

D. RNA nucleotides cannot be easily distinguished from DNA nucleotides

5. Which of the following statement is true concerning the sequence pTACGTAACGTA?

A. This is an RNA sequence

B. In order to have a more rigid structure in a double-stranded molecule, the sequence given would need more adenosine

C. If the complementary strand were present, the number of purines would be equal to the number of pyrimidines

D. The amount of purines is equal to the amount to pyrimidines

6. Complementary base pairing is considered which level of structure?

A. Secondary

B. Tertiary

C. Quaternary

D. Primary

7. Which of the following is/are required for the polymerization reaction catalyzed by DNA polymerase III?

I. RNA primer

II. DNA primer

III. RNA template

IV. DNA template

V. NTPs

VI. dNTPs

VII. Magnesium

VIII. Calcium

A. II, IV, V, and VII

B. II, IV, VI, and VII

C. I, IV, V, and VIII

D. I, IV, VI, and VII

8. DNA polymerase I, from E. coli, has three different catalytic sites. Which of the following is NOT one of them?

A. RNA-dependent RNA polymerase

B. RNA-dependent DNA polymerase

C. DNA-dependent RNA polymerase

D. DNA-dependent DNA polymerase

9. Eukaryotes solve the problem of replicating the ends of their linear chromosomes by:

A. Using a protein as primer

B. Enzyme activity that uses an RNA template and reverse transcription in a series of repeat cycles

C. All of the listed solution

D. Terminal redundancy whereby each end of the chromosome is duplicated allowing recombination

1. Genomic DNA is digested with a restriction enzyme that has a 4 bp recognition (restriction) site. What would be the expected average size of the fragments that would be produced by digesting DNA of random sequence with this enzyme? A) 8 bp B) 16 bp C) 125 bp D) 256 bp E) 512 bp

2.insertion or removal of one or more nucleotide base pairs in DNA within a gene often results in a ____________ mutation. A) transition B) frameshift C) reversion D) transversion E) suppressor

3. Which ingredient is NOT required in Sanger DNA sequencing? A) Primers for DNA polymerase. B) Reverse transcriptase. C) Dideoxy nucleotides. D) Deoxy nucleotides. E) DNA template.

4. What is the purpose of Taq polymerase in a PCR reaction? a. DNA denaturation b. Primer annealing c. DNA synthesis d. Heating of the reaction e. All of the above are associated with Taq polymerase.

5.You are doing lab work with a new species of beetle. You have isolated lines that breed true for either blue shells and long antenna, or green shells and short antenna. Crossing these lines yields F1 progeny with blue shells and long antenna. Crossing F1 progeny with beetles that have green shells and short antenna yield the following progeny: Blue shell, long antenna 92 Green shell, short antenna 88 Blue shell, short antenna 31 Green shell, long antenna 29 Total 240 Assuming that the genes are linked, what is the map distance between them in cM? A) 75.0 cM B) 25.0 cM C) 12.1 cM D) 12.9 cM E) The genes are actually assorting independently. Genes A and B are linked with 10 map units (cM) between them. A heterozygous individual AB/ab (note that the dominants are in coupling) would be expected to produce gametes in which of the following frequencies?

A) 45% AA; 10% Ab; 45% bb

B) 5% AB; 45% Ab; 45% aB; 5% ab

C) 10% AB; 40% Ab; 40% aB; 10% ab

D) 45% AB; 10% Ab; 10% aB; 45% ab

E) 45% AB; 5% Ab; 5% aB; 45% ab

BIOLOGY 172 Lecture Notes - Lecture 21: Shotgun Sequencing, Sanger Sequencing, Dideoxynucleotide

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