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41 Cards in this Set
- Front
- Back
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Restriction enzymes are ....
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part of the microbial immune system; make double stranded breaks at specific sequences in invading DNA & destroy it.
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How to microbes deal with restriction enzymes?
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They modify their DNA so that RE won't recognize them.
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Each restriction enzyme recognizes...
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a specific 4-8 bp pzlindromic sequence.
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There are ____ of restriction enzymes and they are _____ available.
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hundreds; commercially
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Each RE is named for....
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its source
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Microbes have ____ restriction enzymes.
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several different
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How are restriction enzymes used in the lab?
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they are used to cut and paste (ligate) DNAs from different sources. This produces recombinant DNA.
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How do RE's cut the DS DNA?
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They cut them so that they are staggered ends. This makes it so they can be religated to other DNA cuts. They are also palindromic so they will recombine.
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What are the two things that have to occur when DNA is cloned?
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It has to be isolated from its original source and inserted into a vector that can be propagated in a host.
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Vectors are...
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self-replicating DNAs (plasmids or phage) that also contain elements that allow manipulation and/or expression of cloned genes.
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Plasmid insert size and feature.
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1. <20kb
2. replicates independently of microbial chromosome so many copies may be maintained in a single cell. |
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Once DNA are inserted into vectors...
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then the vectors are introduced into host cells.
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Plasmid-derived vectors are introduced by....
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transformation into artifically competent (CaCL2 or electroporation) hosts.
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Phage or viral DNAs are...
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packaged in vitro and then used to infect host cells.
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Bacteriophage insert size and feature.
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1. 9-25 kb
2. packaged into lambda particles; SSDNA viruses have been modified to generate double or single stranded DNA in the host. |
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Cosmid insert size and feature.
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1. 30-47 kb
2. can be packaged into lambda particles for efficient introduction into bacteria, then replicates as a plasmid. |
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PAC's BAC's and YAC's.
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High insert size so almost useless. Artificial chromosomes. Have sequences that work as origins.
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What is the origin of replication in a plasmid used for?
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To maintain high copy number (often 100s in cell) or low as required.
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What is a multiple cloning site?
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It has multiple restriction endonucleases in it that are present only at this site. It is used as a cloning site for genes.
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Because only 1% of cells are competent for plasmid transformation,...
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selectable markers and selective media are used to facilitate screening.
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What are the elements needed in plasmid vectors?
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Origin, multiple cloning site, and selectable markers. There are other elements like gene expression, shuttle vector features, etc.
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Explain the cloning scheme for a vector.
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1. The plasmid is cut with EcoR1 or you insert artifically EcoR1 sides on the gene.
2. Put into test tube at 1:1 molar ration with ligase and ATP. 3. Thie creates a covalent circle DNA. 4. You then plate it and you can sometimes have them seperated by color. 5. Take up the white colonies with a toothpick, let them grow and then analyze them. |
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mRNA cannot be directly cloned, so...
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functional mRNA from eukaryotic cells is first converted to cDNA by using reverse transcriptase.
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Explain the process of making cDNA.
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First you add a primer of all T's to complement a polyA tail. Reverse transcriptase makes the complement strand. Sometimes RNaseH, DNA polymerase, and DNA ligase is needed to finish up.
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The DS-DNA made from the cDNA process can then be cloned by...
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adding artificial RE sites to the ends and inserting into a vector (this is so they can have promoter regiosn etc. to be cloned).
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cDNAs from any source can be expressed as
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proteins when cloned into expression vectors.
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What is a library?
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A collection of DNA. When all DNA from a particular source are simultaneously cloned.
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Explain the DNA library system with an antibody.
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You would take the antibody from the person that you wanted, make a library in a test tube, plate it and see which one binds to the antibodies from that person.
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How can libraries be screened?
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1. hybridization to a labeled NA probe
2. binding to a particular antibody or other ligand after induction of protein expression 3. bioassay |
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PCR stands for
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polymerase chain reaction
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What is PCR?
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You amplify a sequence of DNA 10^6-10^9 times in one round. The most widely used diagnostic technique in biological sciences. Important in diagnostics.
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In PCR some substances inhibit..
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thermophilic DNAPs and give false negatives.
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What is a fairly common problem in PCR?
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False positives because of extreme sensitivity of technique. A single cell's gene can be detected. Also, many outside contaminations can occur.
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Genes that undergo PCR can be...
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cloned, sequenced, & expressed.
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What are primers?
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short SS-DNA (10-30 b) that bind at ends of denatured templates
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The template for PCR can be...
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any source of DNA. And if it is RNA it must undergo reverse transcriptase to get the DNA.
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Replicating DNA occurs at....
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high temps, using DNAPs from thermophiles and dNTPs.
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Currently, PCR reactions are done with...
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thermocyclers, but field versions are being developed.
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How long does 20-30 cycles of PCR take?
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1-3 hours.
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In sequencing DNA, each sequencing machine can sequence _____ per day.
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10^6 basesq
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What is a shotgun procedure?
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When entire genomes are chopped up into smaller pieces, simultaneously sequence them all, and then a computer will find overlapping areas of sequence and put the whole sequence back together.
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