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32 Cards in this Set
- Front
- Back
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What do Helicase enzymes do?
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Unwind a small portion of the double-stranded DNA
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What does DNA polymerase do?
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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
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Can we replicated large-scale DNA replication in vitro?
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No
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How long are the DNA strands that we can replicate?
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Several milion-fold
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What is used to separate the DNA strands, (denature)?
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Heat
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What are primers?
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Chemically synthesized, 20-30 nucleotide long pieces of single-stranded DNA that are desgined by the researchres to amplify a specific region of DNA.
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How do primers work?
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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
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What end of both primers must face the target DNA?
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3', because of the anti-parallel nature of DNA
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What is annealing?
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Primer binding
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What is extension?
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DNA polymerization
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How many times is the cycle of denaturation, annealing, and extension repeated?
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15-35 times
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Who came up with the idea for PCR?
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Kary Mullis
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Why did Mullis have to use new enzymes for each cycle of the reaction?
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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)
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From which species do we get the DNA polymerase now?
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Thermus aquaticus, a thermophilic bacterium, which lives in the thermal pools of Yellowstone National Park
Taq polymerase |
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What is mixed together for PCR?
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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
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What are the typical temperatures of DNA polyermase?
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30 seconds at 94º ~ denature
30 seconds at 55º ~ anneal, (hybridize) 30 seconds at 72º ~ Taq extends the primers |
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Why does the template DNA sanneal to the primers and not the original double helix?
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Because the primers are present at much higher concentrations that the template DNA
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How many cycles does it take to have an amplificiation factor of one billion?
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33
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How many factors does the number DNA sequences increase by in each cycle?
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It doubles
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What does gel electrohporesis do overall?
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Evaluates the PCR product.
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How does DNA acquire a net negative charge?
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In inoic solution, the acidic phosphate backbone of DNA liberates hydrogen ions and gives the DNA molecule net negative charge
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What are two sieve-like substances in the gel?
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Agarose or acrylamide
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What is the gel immersed in after DNA is loaded into the depressions?
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A buffered ionic solution
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What is the gel stained with?
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Ethidium bromide or SYBR Green
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What is a polymorphism?
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A characteristic that differs among different members of a population.
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Where do most polymorphisms occur?
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In non-coding DNA and do not affect a polypeptide product
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How must polymorphisms be detected?
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By sequencing, PCR, or restriction enzyme digest
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What is the most common type of polymorphism?
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Singlenucleotide polymorphism, (SNP)
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How many alleles do most NSPs have?
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2 alleles
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What is a repeat polymorphism?
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a short sequence that is repeated head-t-tail a variable number of times
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What is the relationship between the number of times the sequence is repeated and the PCR product?
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The more times the sequence is repeated, the longer the PCR product
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What CEPH cell lines?
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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.
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