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32 Cards in this Set

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  • Back
What do Helicase enzymes do?
Unwind a small portion of the double-stranded DNA
What does DNA polymerase do?
Binds to the DNA/RNA heteroduplezs and moves along the template DNA strand, polymerizing a strand of complementary DNa while other enzymes remove the NRa portion fo the heteroduplexes
Can we replicated large-scale DNA replication in vitro?
How long are the DNA strands that we can replicate?
Several milion-fold
What is used to separate the DNA strands, (denature)?
What are primers?
Chemically synthesized, 20-30 nucleotide long pieces of single-stranded DNA that are desgined by the researchres to amplify a specific region of DNA.
How do primers work?
One primer is complementary to a sequence on one side of the target DNa, the other primer iscomplementary to sequence on the other side of the target DNA; DNA polymeraseadds nucleotides to the 3' end of the primers, doubling the number of target DNA sequences
What end of both primers must face the target DNA?
3', because of the anti-parallel nature of DNA
What is annealing?
Primer binding
What is extension?
DNA polymerization
How many times is the cycle of denaturation, annealing, and extension repeated?
15-35 times
Who came up with the idea for PCR?
Kary Mullis
Why did Mullis have to use new enzymes for each cycle of the reaction?
She used E. coli DNa polymerase, which denatures at the same temperature used to separate the DNA strands; (she also neeed to move the tubes to a different water both for every temp. change)
From which species do we get the DNA polymerase now?
Thermus aquaticus, a thermophilic bacterium, which lives in the thermal pools of Yellowstone National Park

Taq polymerase
What is mixed together for PCR?
A small quantity of template DNA, buffer, Taq DNA polymerase, two oligonucleotide primers, the four deoxynucleotides building blocks of NDA, (dATP, dCTP, d GTP, dTTP), and the cofactor MgCl2
What are the typical temperatures of DNA polyermase?
30 seconds at 94º ~ denature

30 seconds at 55º ~ anneal, (hybridize)

30 seconds at 72º ~ Taq extends the primers
Why does the template DNA sanneal to the primers and not the original double helix?
Because the primers are present at much higher concentrations that the template DNA
How many cycles does it take to have an amplificiation factor of one billion?
How many factors does the number DNA sequences increase by in each cycle?
It doubles
What does gel electrohporesis do overall?
Evaluates the PCR product.
How does DNA acquire a net negative charge?
In inoic solution, the acidic phosphate backbone of DNA liberates hydrogen ions and gives the DNA molecule net negative charge
What are two sieve-like substances in the gel?
Agarose or acrylamide
What is the gel immersed in after DNA is loaded into the depressions?
A buffered ionic solution
What is the gel stained with?
Ethidium bromide or SYBR Green
What is a polymorphism?
A characteristic that differs among different members of a population.
Where do most polymorphisms occur?
In non-coding DNA and do not affect a polypeptide product
How must polymorphisms be detected?
By sequencing, PCR, or restriction enzyme digest
What is the most common type of polymorphism?
Singlenucleotide polymorphism, (SNP)
How many alleles do most NSPs have?
2 alleles
What is a repeat polymorphism?
a short sequence that is repeated head-t-tail a variable number of times
What is the relationship between the number of times the sequence is repeated and the PCR product?
The more times the sequence is repeated, the longer the PCR product
What CEPH cell lines?
Cell lines generated by collecting lymphocytes , (present in the blood), and infecting them with Epstein-Barr virus. The virus immortalizes the cells so that they will continue to divide indefinitely.