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50 Cards in this Set
- Front
- Back
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Molecular cloning
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Cloning of pieces of DNA or genes
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Cloning: Step 1
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Restriction Endonuclease
Method for cutting DNA at precise locations |
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Cloning: Step 2
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DNA Ligase
Method for covalently joining two DNA molecules |
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Cloning: Step 3
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Transformation, infection, electroporation
Procedure for moving recombinant DNA from the test tube into the host |
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Cloning: Step 4
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DNA vector
Mechanism of carrying and replicating segments of DNA within a host organism |
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Cloning: Step 5
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Methods to select and identify those host cells harboring desired recombinant DNA molecules
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What are the two different types of ends made by restriction endonucleases?
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Blunt ends
Cohesive ends |
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Why are cohesive ends easier to join? What is the caveat?
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Cohesive ends are held together by base pairing.
The condition is that the overhangs must be compatible. |
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How do we generate a recombinant molecule?
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1) Cleave DNA to generate restriction fragment of interest.
2) Cleave vector with compatible enzyme. 3) Ligate the DNA molecules |
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What is the restriction fragment?
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The fragment of the chromosomal DNA that's cut with restriction endonucleases.
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What enzymes joins the fragments? What is the energy cost?
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DNA ligase, an ATP for each side
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How do polylinker sites facilitate cloning?
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They provide many different sites each recognizable by a different endonuclease
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What are the three different possible cloning vectors in the order of increasing DNA fragment cpacity?
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Plasmid, bacteriophage lamba and BACs
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How are plasmid introduced?
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Transformation
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How are bacteriophages introduced?
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Phage infection
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How are BACs introduced?
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Electroporation
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Characteristic of a typical plasmid vector (pBR322)
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1)Origin of replication to allow for replication in the host.
2) Unique restriction sites 3) Selectable markers that enable bacteria harboring plasmid to survive |
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What are two types of selectable markers?
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Ampicillin and tetracycline resistance
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Transformation
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Calcium chloride results in the inefficient enterance of plasmid
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What is the role of the lamba arms of the bacteriopahage?
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Essential for phage propagation:
Packing Making the phage head |
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What enzyme cleaves X-gal?
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B-Galactosidase
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Replica Plating
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Allows test on two different resistance marker
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Library
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Collection of DNA fragments each carried in a vector
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Genomic Library
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Contains organism specific, cell-type independent genomic DNA
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cDNA Library
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Contains organism specific, cell-type specific DNA copies of mRNA
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What enzyme is used for cDNA?
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Reverse Transcriptase
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What enzymes are used to degrade the RNA template in making cDNA?
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RNase H
Alkali Hydrolysis |
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Why can we digest RNA with Alkali Hydroylsis and not DNA?
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2 'OH is used to 2', 3' cyclic phosphate bond.
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Type of probes
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homologous
mRNA isolate designed probe from protein synthesis amplfy probe with PCR |
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How do we gene sequencing?
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generate fragments: dideoxy sequencing which ddNTP are chain termininating because they don't have a 3' OH
nested fragmenets are sorted based on size using capiliary gel-electrophoresis |
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What are the limits of dideoxy seqeuncing?
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up to 600 base pairs
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What percentage DNA is translated to protein?
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1 to 1.4 %
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What percentage of DNA is transcribed to RNA?
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25 %
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What is the key to PCR?
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Thermostable RNA polymerase
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What are repeated steps of PCR?
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Seperate strands by applying heat
Hybridize primers complementary to the desired genes Polymerize DNA copy |
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Application of PCR
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Amply the number of restriction sites
Identifying STRs |
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What are STR?
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Loci that are common
Number of repeats vary among individuals Short Tandem Repeat Allow us generate a unique genetic fingerprint by using multiple alleles Fragments of different lengths based on the individual that it came from. |
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What does Site-Directed Mutagenesis allow us to do?
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Introduce mutations in our clone
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How is Site-Directed Mutagenesis accomplished?
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Attachment of oligonucleotide with the the desire sequence change to a single strand recombinant DNA.
Use DNA polymerase to copy strand so that one of the strands carries the mutation. |
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What do we want to avoid in designing probes?
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Min. degeneracy
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What do expression vectors allow us to do?
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They allow us to create recombinant proteins from a given gene.
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What are important features of expression vectors?
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Circular plasmid
Origin of replication Selectable markers Ribosome binding site Transcription termination sequence Genes encoding repressor |
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IPTG
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Inducer for lac operon
Not metabolismable allolactose |
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Protein tags
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Facilitate purification of recombinant protein through chromatography
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How do we utilize protein tags?
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Gene for target protein is fused with gene for protein tag (GST) which binds to glutathione
Transcription and translation Cell extract containing fusion protein is removed Extract is purified using a column that anchors to the protein tag (contains gluthathione) Once all no desired proteins are eluted with various salts, wash with glutathione tag to remove the fusion proteins |
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Yeast 2-Hybrid Analysis
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Fusion protein is used to understand protein-protein interactions.
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How does yeast 2-hybrid analysis work?
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Fuse protein X with DNA binding domain
Fuse another protein Y with the activation domain If X and Y interact, this leads to transcription. |
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What is the common protein used for yeast 2 hybrid analysis?
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Gal4p
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DNA microarray
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Use a library on chip to study gene expression.
What gene are on at some point? |
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How is DNA microarray analysis performed?
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Isolate mRNA, make cDNA from each stage, label differently, the chip carries either cDNA or genomic library, hybridize, if gene is on in both situation then both with marked, if gene is on a specific time, only one of the cDNA from the the stages will be maked
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