Term
| Describe the two steps of Cloning gene |
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Definition
1. inserting gene into self-replicating plasmid or virus
2. amplifying
-usually in E. col
-PCR |
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Term
| Explain the role of endonucleases in the insertion process |
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Definition
1. cuts DNA at specific sequences
-4-6 bp palindromes
2. two types
a. blunt: cut straight through- no lining up to bind
b. sticky: creates overhang that binds right back to same sequence
3. endonucleases are named after the bacteria that make them
-used in the protection against viruses |
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Term
| How do bacteria prevent endonucleases from cutting their own DNA? |
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Definition
-Bacterial DNA is methylated blocking the function of endonucleases
-the endonuclease binds on both sides and makes cut between same nucleotides (sticky)
-methylation blocks the enuc from binding |
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Term
Describe the following endonucleases
1. EcoRI
2. HincII
3. HindIII
4. HpaII
5. AluI |
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Definition
1. E. coli - sticky
2. Haem. influ. - blunt
3. Haem. influ. - sticky
4. Haem. parainflu. - sticky
5. Arthro. luteus - blunt |
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Term
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Definition
1. Must contain an origin of replication
-OriC or ARS
2. Dominant selectable marker
-something that confirms insertion of the gene
ex. green fluorescent protein or antibiotic resistance
3. unique restriction sites |
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Term
| Describe examples of cloning vectors |
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Definition
1. Plasmids - 5-10kb
2. Bacteriophage - 10-15kb
3. Cosmid- 35-45kb
-contain plasmid and bacteriophage DNA
-OriC and has efficient transport of virus
4. Shuttle - replicate in eu and pro (OriC and ARS)
5. YAC (yeast artificial chormosome)- 200kb |
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Term
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Definition
1. in vitro cloning technique that requires knowledge of flanking sequence
2. not useful for long products (>35 kb)
3. 3 stages
a. denature- pull apart strands
b. anneal- bind polymerase
c. extension- polymerase lays down bases |
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Term
Describe DNA libraries
1. Genomic
2. cDNA
3. Uses |
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Definition
1. entire genome
-sticky cutting: allows for precise excision
-blunt: ends must be added using terminal transferase to 3' end (adds Ts or As)
2. only coding DNA sequences
-Oligo T's added to end
-reverse transcriptase creates complimentary strand
-second cDNA strand is synthesized
3. Complementation screening
-in situ hybridization
-site directed mutagenesis |
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Term
Describe the following tests
1. Western
2. Southern
3. Northern |
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Definition
1. run proteins and then add antibodies to detect presence
2. run DNA in gel, transfer to paper, add ssDNA to bind and label
3. run RNA, add ssDNA (degrades quickly) |
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Term
| Describe restriction sites and sequencing |
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Definition
-PCR is set up with poison nucleotides added
-ddGTP does not contain 3'OH so polymerase will add it and then it will not allow for additional polymerization
-run through acrylamide gel and differences in length can determine sequence |
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