Term
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Definition
1. Digestion of DNA
2. Digestion of vector (w same restriction enzyme)
3. Ligation
4. Transformation/transfection
5. Finding colony with recombinant DNA
-and then if it's the DNA you're after, just use the same restriction enzymes after cells are replicated |
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Term
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Definition
| -Process of introducing an exogenous gene – called a transgene – into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. (such as in mice) |
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Term
| What are four types of vectors and the bp length they are useful for? |
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Definition
1. Plasmids - 10-16 kbp
2. Bacteriophage λ DNA - Up to 23 kbp
-replicate DNA in bacteria
3. Cosmids - Up to 45 kbp
-from λ DNA COS sites + plasMIDS
4. YACs (yeast artificial chromes) - 100-3000 kbps (ie. whole human chromosomes or substantial parts of them)
-the other three are maintained in E. coli |
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Term
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Definition
-A type of cloning vector that can accept DNA but also has the necessary hardware in the correct places for expression of a desirable protein
-has promotor, terminator, Shine Dalgarno sequence, usually repressors are removed, etc. |
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Term
| Three things YACs must have? |
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Definition
| -CEN (centromere), TEL (telomere), ARS (autonomously replicating sequence - contains origin of replication) |
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Term
| How do we identify cells transformed with YACs? |
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Definition
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Term
| Advantages to using YACs? |
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Definition
-Yeast can do post translational modification
-No endotoxins attached like in bacteria (lower activity) |
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Term
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Definition
-Collection of clones that contain all of the cDNA corresponding to the genes expressed in particular tissue
-Source of DNA is mRNA (requires reverse transcriptase)
-cDNA represents sequences expressed as RNA |
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Term
| What enzymes are needed to make a cDNA library? |
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Definition
-Reverse transcriptase
-RNase H - degrades RNA template (can also use an alkaline solution)
-DNA pol 1 (usually replaces primers)
-S1 Nuclease - cuts loop that forms (used at end)
-Terminal transferase - fixes 3' overhang |
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Term
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Definition
-Defective RNA component of telomerase (does the overlap)
-Abnormal nails, white patches in mouth, lacy rash on face, neck, and chest |
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Term
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Definition
1. Digest DNA
2. Agrose gel electrophoresis
3. Denature in alkaline solution
4. Blot/transfer ssDNA onto nitrocellulose membranes
5. Hybridize with labeled probe
-Involves prehybridization, hybridization, and washing under either high stringency (low salt high temp) or low stringency (high salt low temp) conditions
6. Find bands (autoradiography)
-overall it is used to find homology in DNA using a probe |
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Term
| Single gene probe vs allele specific probe ASP? |
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Definition
-The ASP is shorter and extremely specific (so one must know the specific mutation to design and use)
-With single gene probe we will usually use low and then high stringency conditions to show levels of homology
-We can use ASP in DOT BLOTTING |
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Term
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Definition
-Uses ASP to quickly identify disease
-We obtain, denature, and transfer DNA to nitrocellulose membrane (may or may not need PCR)
-Then we treat with two kinds of ASPs, one for the disease and one for normal allele, and wash
-More useful for small sequence changes |
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Term
| What enzyme does PCR rely on? |
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Definition
| -Taq polymerase, because it can operate at the increased temperatures reached during temp cycling |
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Term
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Definition
-Reverse transcriptase can be used in conjunction with PCR to amplify only c-DNA
-Especially useful for finding levels of circulating HIV |
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Term
| Sanger's chain termination DNA sequencing? |
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Definition
-Uses tweaked levels of dNTPs and labeled ddNTPs to sequence DNA strands
-The ddNTPs lack the 3' OH and terminate chain extension
-They are infrequently incorporated
-Traditionally run with four separate reactions (but now we have different colors for each ddNTP) |
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Term
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Definition
-Restriction fragment length polymorphism
-Basically just uses restriction enzymes on sample DNA and looks at what fragment lengths are attained by using gel electrophoresis
-Can be used to test for diseases such as sickle cell, paternity testing, etc.
-note; PCR alone can also be used for paternity testing by using primers specific to hypervariable microsatellite sequences |
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Term
What is analyzed and probe used in;
-Southern
-Northern
-Western
-ELISA |
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Definition
-DNA, oligonucleotide
-mRNA, oligonucleotide (RNA)
-Protein, antibody
-Protein or antibody, antibody or protein |
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Term
| Type I, II, & III endonucleases? |
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Definition
-I cuts randomly at 1000+bp away
-II cuts in palindromic sequence
-III cuts 25bp after |
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Term
| What is happening at the temperatures of PCR? |
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Definition
-95; denaturing
-50; annealing of primers
-72; polymerizing |
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