Term
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Definition
1.) Identify gene associated with a mutant phenotype.
2.) Predict function from amino acid sequence
3.) Develop specific antibodies from large amounts of recombinant proteins
4.) Make mutants and test effects on function
5.) Commercial purposes and gene therapy |
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Term
| Step 1 in Cloning Genomic DNA |
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Definition
| Isolate a fragment of DNA from organism using restriction enzymes to cut DNA at specific sequences and using ligase to join the fragments |
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Term
| Step 2 in Cloning Genomic DNA |
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Definition
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Term
| Step 3 in Cloning Genomic DNA |
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Definition
Transform the host cells with the cloning vector
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Term
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Definition
Function: Cut phosphodiester bonds of DNA within a DNA molecule - Each cuts a specific DNA sequence, usually palindromic
- Cuts in a staggered way
- Ends of DNA have a 5'-PO4 and 3'-OH
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Term
| Restriction Endonuclease Naming |
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Definition
Names come from bacterial names: - EcoRI (RI): from Escherichia Coli
- RsaI (R): from Rhodopseudomonas sphaeroides
- BamHI (B): from Bacillus amyloliquefaciens
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Term
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Definition
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Term
| What do cloning vectors have in common? |
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Definition
- Origin of replication
- Selectable marker
- Multiple restriction sites for cloning
- High copy number
- Most are used in unicellular hosts
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Term
| Essential and Useful features of bacterial plasmids: |
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Definition
- Essential:
- Origin of replication
- Carry dominant selectable marker
- Use unique restriction enzyme sites in plasmid to clone ineo
- Useful:
- Polylinker with multiple unique restriction enzyme sites
- May have visible marker
- High copy number
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Term
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Definition
- One of the first bacterial plasmids engineered for cloning
- p=plasmid, UC=university of California
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Term
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Definition
| If lacZ is intact, there is a blue pigment; if it is not, there is no pigment (white) |
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Term
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Definition
A collection of clones containing DNA fragments from an entire genome - Often made using partially digested DNA
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Term
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Definition
Collection of clones containing DNA fragments representing all mRNAs in particular cell type or under specific conditions |
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Term
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Definition
- Used for purigying mRNAs and for priming DNA synthesis
- Anneals to polyA tail
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Term
| Reverse Transcriptase (RT) |
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Definition
- RNA dependent DNA polymerase
- Makes complementary DNA copy of mRNA called cDNA
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Term
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Definition
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Term
| Components in cDNA Library Construction |
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Definition
- oligo-dT: selects mRNA to use
- Reverse Transcriptase: converts to ssDNA
- RNase H: degrades mRNA leaving small RNA primers
- DNA polymerase I: creates 2nd strand
- Ligase: links fragments of second strand
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Term
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Definition
| Links to ds cDNA with the aid of DNA ligase to allow for insertion into vector |
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Term
Vector: Plasmid - Form of vector?
- Host?
- Ave. insert size?
- Major uses?
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Definition
- Ds circular DNA
- E. coli
- < 10
- Cloning specific DNA fragments
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Term
Vector: BAC - Form of vector?
- Host?
- Ave. insert size?
- Major uses?
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Definition
- Bacterial artificial chromosome
- E.coli
- 200
- Genomic libraries and genome sequencing projects
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Term
Vector: YAC - Form of vector?
- Host?
- Ave. insert size?
- Major uses?
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Definition
- Yeast artificial chromosome
- Yeast
- 200-500
- Genomic libraries and genome sequencing projects
- Not widely used because sequences are often rearranged
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Term
| Process of finding the clone of interest by using antibody in a fusion protein: |
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Definition
- Digest DNA with EcoRI
- Ligate cDNA with EcoRI linker into host DNA
- Plate on bacterial lawn
- Overlay membrane
- Remove membrane; protein binds to membrane
- Incubate membrane with primary antibody, wash, incubate with secondary antibody
- Fusion protein bound to membranes
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Term
| Process of finding specific nucleic acids by using gel electrophoresis and blotting: |
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Definition
- Digest to completion the genomic DNA with a restriction enzyme and subject the digest to agarose gel electrophoresis
- Transfer DNA to a membrane filter by "wicking"
- Mix with probe in sealed plastic bag, typically overnight
- Dry and expose to X-ray film
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Term
ddNTPs What does it stand for and what process is it a part of? |
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Definition
| This stands for dideoxynucleoside triphosphate, and it is used in the chain termination/Sanger method. |
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Term
| Process of Sequencing by the Sanger Method: |
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Definition
- ss DNA template is primed for DNA synthesis; all synthesized strands start at same site
- 4 ddNTPs are added to strands of DNA at each Adenine; cannot form phosphodiester bonds
- The addition of dNTPs causes the strand to stop replicating
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Term
| Description of the polymerase chain reaction: |
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Definition
| A very powerful method to isolate genes and detect mutations. It is a sequential series of DNA synthesis reactions using forward and reverse primers on each side of the sequence to be amplified: denature, anneal primers, chain extend, and repeat. |
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Term
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Definition
- Heat to separate strands
- Add synthetic oligo-nucleotide primers; cool.
- Add heat-tolerant DNA polymerase to catalyze 5'-->3' DNA synthesis
- Repeat steps 1 and 2
- DNA synthesis is catalyzed by the heat-tolerant DNA polymerase
- Repeat steps 1 through 3
- After 25 cycles, the target sequence has been amplified about 10^6-fold
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