1|Page
Cell Bio Exam 3 Dimario, chapter
6 Exam 100% Solved Graded A+
What is Polymerase Chain Reaction? Ans: PCR amplifies specific DNA
exponentially
- most powerful
How does PCR work? Ans: Forward and Reverse primers select the specific
sequence of DNA to be amplified (Primer is an oligonucleotide)
in vitro organic synthesis is backwards using Ans: a 5' OH
In PCR denaturation occurs at ? Ans: 95
In PCR annealing of primers occurs at? Ans: 55
Elongation of primers occurs at? special heat stable dna polymerase Ans: 72
first cycle in PCR depends on? Ans: Tm of the primers
How is the primer able to attach to the DNA without the single strands
reannealing to each other? Ans: Primers are in vast molar excess compared to
the original template DNA (they serve as starting points); this allows for
multiple cycles
,2|Page
Why would you design your primers to include restriction enzyme sites? Ans:
allows you to ligate with to a plasmid vector with the same restriction sites.
What is directional ligation? Ans: ligate with plasmid with sticky ends after cut
with restriction enzymes
What is A Newer Method for Producing Recombinant Plasmids: Ans: gipson
assembly
gipson assembly ligates Ans: several PCR products all at once in the correct
orientation
Gipson assembly anneals Ans: compatible sticky ends
- 3 extension with a polymerase to fill in gaps
What is Maxam-Gilbert Sequencing? Ans: end labeling DNA fragments, then
chemically cleaving the DNA
Maxam and gilbert dna sequencing technique uses what end labeling? and
what material? Ans: 32P added to 5' ends by a kinase
- Polyacrylamide gel/autoradiography
Sanger sequencing uses? and sequences how much? Ans: dideoxy-
ribonucleosides triphosphates (ddNTPs)
- up to 500-600 bases per run
, 3|Page
No 3' hydroxyl means? Ans: no further synthesis (gel termination)
What is Sanger sequencing? Ans: A technique to determine the sequence of
DNA, that uses dideoxynucleotides to prevent further extension past a known
base.
- Stops at the dideoxynucleotide because it does not contain a 3'-OH to
continue synthesizing.
- Primer (oligonucleotide) is 5' end-labeled with 32P
- produces many labeled fragments of different sizes
What is bioinformatics? Ans: Revolution in computer technology to handle
the vast sequence information.
How does a computer process and sequence a genome from a Sanger
sequence? Ans: A computer detects the fluorescent nucleotides in real time
Explain the process of Next Generation DNA Sequencing. Ans: 1.) Linkers are
ligated to ends of genomic DNA fragments (very short, ~100bps)
2.) Perform PCR, but with both primers attached to the solid supports (primers
annealed to linker DNA)
3.) This leaves you with ~1000 identical DNA fragments tethered to the solid
support but in a very tight micro-cluster, a "colony" of identical fragments
Cell Bio Exam 3 Dimario, chapter
6 Exam 100% Solved Graded A+
What is Polymerase Chain Reaction? Ans: PCR amplifies specific DNA
exponentially
- most powerful
How does PCR work? Ans: Forward and Reverse primers select the specific
sequence of DNA to be amplified (Primer is an oligonucleotide)
in vitro organic synthesis is backwards using Ans: a 5' OH
In PCR denaturation occurs at ? Ans: 95
In PCR annealing of primers occurs at? Ans: 55
Elongation of primers occurs at? special heat stable dna polymerase Ans: 72
first cycle in PCR depends on? Ans: Tm of the primers
How is the primer able to attach to the DNA without the single strands
reannealing to each other? Ans: Primers are in vast molar excess compared to
the original template DNA (they serve as starting points); this allows for
multiple cycles
,2|Page
Why would you design your primers to include restriction enzyme sites? Ans:
allows you to ligate with to a plasmid vector with the same restriction sites.
What is directional ligation? Ans: ligate with plasmid with sticky ends after cut
with restriction enzymes
What is A Newer Method for Producing Recombinant Plasmids: Ans: gipson
assembly
gipson assembly ligates Ans: several PCR products all at once in the correct
orientation
Gipson assembly anneals Ans: compatible sticky ends
- 3 extension with a polymerase to fill in gaps
What is Maxam-Gilbert Sequencing? Ans: end labeling DNA fragments, then
chemically cleaving the DNA
Maxam and gilbert dna sequencing technique uses what end labeling? and
what material? Ans: 32P added to 5' ends by a kinase
- Polyacrylamide gel/autoradiography
Sanger sequencing uses? and sequences how much? Ans: dideoxy-
ribonucleosides triphosphates (ddNTPs)
- up to 500-600 bases per run
, 3|Page
No 3' hydroxyl means? Ans: no further synthesis (gel termination)
What is Sanger sequencing? Ans: A technique to determine the sequence of
DNA, that uses dideoxynucleotides to prevent further extension past a known
base.
- Stops at the dideoxynucleotide because it does not contain a 3'-OH to
continue synthesizing.
- Primer (oligonucleotide) is 5' end-labeled with 32P
- produces many labeled fragments of different sizes
What is bioinformatics? Ans: Revolution in computer technology to handle
the vast sequence information.
How does a computer process and sequence a genome from a Sanger
sequence? Ans: A computer detects the fluorescent nucleotides in real time
Explain the process of Next Generation DNA Sequencing. Ans: 1.) Linkers are
ligated to ends of genomic DNA fragments (very short, ~100bps)
2.) Perform PCR, but with both primers attached to the solid supports (primers
annealed to linker DNA)
3.) This leaves you with ~1000 identical DNA fragments tethered to the solid
support but in a very tight micro-cluster, a "colony" of identical fragments
