USCSOM: Biochemistry: L36-38 Recombinant DNA I&II
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| What is cDNA? | DNA derived from copying RNA
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| Restriction enzymes tend to leave what on the 3' end and what on the 5' end? | 3'OH and 5'PO4
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| What kind of primer is generally used on mRNA in reverse transcriptase? | oligo dT; short oligonucleotides of pure T
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| What kind of gel is used to separate molecules from a few nts up to a few thousand nt in length? | Polyacramide gel
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| What separation technique is used for bases between 100nt to 20,000nt? | Agarose gel
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| What technique is use dto separate fragments up to 1 million bps? | pulsed field gel electrophoresis
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| What kind of stain is used to visualize DNA fragments from electrophoresis? | ethidium bromide
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| What kind of blot test is used to probe specific DNA fragments? | Southern blots
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| What kind of blot test is used to probe specific RNA fragmnts? | Northern blots
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| What kind of blot test uses antibodies to probe proteins? | Western blots
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| What are hybridization arrays? Advantages? | micro-dot arrays of short oligonucleotides; allow testing on many mRNAs simultaneously
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| Describe the Sanger method. | requires a primer, DNA polymerase, and a dideoxy terminator; all four ddNTPs are put on a gel and read from the bottom up
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| What is a vector? | host/carrier DNA into which foreign DNA is grafted
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| What are two types of vectors? | plasmids and viruses
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| What are the characteristics of plasmids as vectors? | easy to work with, good for small DNA fragments
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| Why would you use viruses for a vector? | larger amounts of DNA; more efficient at infecting cells
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| What are YAC Vectors? | yeast artificial chromosomes; hold huge amounts of DNA; awkward to work with and maintain
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| What are BAC vectors? | bacterial artificial chromosome; based on E. coli; fragments of 300kb; current vector of choice
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| What is pUC18? | cloning vector used in E. coli; small circular ds DNA that replicates in E. coli
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| What is an expression vector? | designed to insert cDNAs downstream of a bacterial mormoter
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| How do you screen cDNA libraries? | specific antibodies
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| What is PCR? | polymerase chain reaction
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| How many bps are the primers in PCR? | 20bps; long enough for a unique sequence
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| What is the general bp limit to amply a sequence via PCR? | 10 kb
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| What is a common thermostable DNA polymerase used in PCR? | Taq polymerase
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| What is a single nucleotide polymorphism? | single base pair change that does not code for anything
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| What is a restriction fragment length polymorphism? | mutation that creates or abolishes the recognition site for a restriction enzyme
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