PCR and DNA Sequencing Exam Questions and Answers Already Passed
Polymerase Chain Reaction (PCR) - Answers Is very fluid and changing; always new tech.
Developed in the 1980s. Reiterative DNA synthesis in vitro using specific oligonucleotide primer pairs.
Short stretch of nucleotide, usually 20 nucleotides long used as primers for DNA synthesis.
exponential amplification of a specific genomic DNA fragment. MANY MANY copies.
Tube requirements - Answers Template DNA (any type of DNA)
DNA polymerase
dNTP's (dATP, dCTP, dGTP)
Buffer, right amount of salt
A pair of single stranded Oligonucleotide Primers that hybridize to a specific genomic locus (position)
need pairs. (5' to 3') and (3' to 5').
Reaction requirements - Answers the reaction is run for many cycles, each cycle has three steps:
Denature 95C
Anneal 60C
Polymerization 72C
Typically run for 30-40 cycles
Denature - Answers separate the strands.
, anneal primers - Answers Lower temp to create H-bonds between primers and template strands.
In labs primers don't have to be RNA, they can be chemically synthesized DNA.
Note position and orientation of primers.
DNA polymerization - Answers Taq pol + dNTPs @ 70C to create, polymerize new strands.
Taq polymerizes 5' --> 3' and requires a 3'- OH (from oligonucleotide primer).
1 full cycle of PCR - Answers You end up with 2-double stranded DNA molecules.
Thermocycler - Answers Is a PCR machine which allows amplification to proceed through successive
rounds. It raises and lowers the temperature. In order to denature, anneal, and polymerize.
Can start with small # of templates and amplify until you have more than a million molecules of DNA.
(2^40)l
To calculate amplification - Answers 2^n= double-stranded DNA segments (where n=# of cycles).
Clicker question 1: - Answers What DNA polymerase properties would you want if you could design the
perfect polymerase for PCR reactions?
A. 3' --> 5' proofreading activity --> want DNA sequence to be as accurate as possible.
C. Able to maintain activity at high temperatures --> DNA polymerase is denatured at high temps, needs
to be effective at 90C.
Taq polymerase - Answers Heat denaturation of template doesn't inactive the enzyme activity of Taq.
What makes PCR special - Answers It has been revolutionary b/c of its ease, speed, and sensitivity.
By picking primers it allows for the isolation and amplification of specific DNA fragments.
Application of PCR - Answers Detection of genetic variation for:
Forensics (DNA fingerprinting)
Medical genetic diagnostics
Polymerase Chain Reaction (PCR) - Answers Is very fluid and changing; always new tech.
Developed in the 1980s. Reiterative DNA synthesis in vitro using specific oligonucleotide primer pairs.
Short stretch of nucleotide, usually 20 nucleotides long used as primers for DNA synthesis.
exponential amplification of a specific genomic DNA fragment. MANY MANY copies.
Tube requirements - Answers Template DNA (any type of DNA)
DNA polymerase
dNTP's (dATP, dCTP, dGTP)
Buffer, right amount of salt
A pair of single stranded Oligonucleotide Primers that hybridize to a specific genomic locus (position)
need pairs. (5' to 3') and (3' to 5').
Reaction requirements - Answers the reaction is run for many cycles, each cycle has three steps:
Denature 95C
Anneal 60C
Polymerization 72C
Typically run for 30-40 cycles
Denature - Answers separate the strands.
, anneal primers - Answers Lower temp to create H-bonds between primers and template strands.
In labs primers don't have to be RNA, they can be chemically synthesized DNA.
Note position and orientation of primers.
DNA polymerization - Answers Taq pol + dNTPs @ 70C to create, polymerize new strands.
Taq polymerizes 5' --> 3' and requires a 3'- OH (from oligonucleotide primer).
1 full cycle of PCR - Answers You end up with 2-double stranded DNA molecules.
Thermocycler - Answers Is a PCR machine which allows amplification to proceed through successive
rounds. It raises and lowers the temperature. In order to denature, anneal, and polymerize.
Can start with small # of templates and amplify until you have more than a million molecules of DNA.
(2^40)l
To calculate amplification - Answers 2^n= double-stranded DNA segments (where n=# of cycles).
Clicker question 1: - Answers What DNA polymerase properties would you want if you could design the
perfect polymerase for PCR reactions?
A. 3' --> 5' proofreading activity --> want DNA sequence to be as accurate as possible.
C. Able to maintain activity at high temperatures --> DNA polymerase is denatured at high temps, needs
to be effective at 90C.
Taq polymerase - Answers Heat denaturation of template doesn't inactive the enzyme activity of Taq.
What makes PCR special - Answers It has been revolutionary b/c of its ease, speed, and sensitivity.
By picking primers it allows for the isolation and amplification of specific DNA fragments.
Application of PCR - Answers Detection of genetic variation for:
Forensics (DNA fingerprinting)
Medical genetic diagnostics
