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Definition
| studied directly in the organism without removing |
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| a DNA sequence capable of autonomous replication |
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Name the various transfecting procedures |
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Definition
transfect is same as transform, but usually refers to viral vectors Cap: incubation with a calcium-phosphate solution Electroporation: electric shock Lipofection: incubation with lipids |
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Definition
| totally undifferentiated stem cells that are not committed to any developmental pathway. ES cells |
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| a reagent that is capable of recognizing a desired sequence (epitope) in a complex mixture of many different sequences. |
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Definition
oligo short single-stranded DNA molecule |
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| a collection of DNA clones |
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| Define restriction fragment length polymorphism |
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Definition
RFLP variations in the lengths of restriction fragments VNTR: variable number of tandem repeats |
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Definition
| technology for manipulating genes in the whole animal |
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Definition
| a whole organism in which a specific gene has been disrupted so as to remove that gene function in the organism |
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| Describe the function/significance of restriction endonucleases |
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Definition
they recognize groups of bases (4-8) in a specific manner THey make double-stranded breaks they allow highly specific maps of organismal genomes to be made sequences are arranged in palindromes they usually create sticky ends, which are important in the cloning of foreign DNA Ex: EcoRI recognizes: 3 GAATTC 5 5 CTTAAG 3 cleaves after the G (going from 3-5 end) |
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Term
List different types of vectors |
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Definition
Plasmid: circular DNA molecule that can replicate in bacteria Cosmid: vector based on bacteriophage λ BAC: bacterial artificial chromosome (F-factor plasmid) PAC: P1 artificial chromosome (P1 bacteriophage) YAC: yeast artificial chromosome |
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| Describe the process in which RT-PCR is used to clone a gene of interest |
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Definition
the mRNA transcript for that particular gene can be isolated and undergo RTPCR to form cDNA molecules. This eliminates all unexpressed genes. - mRNA transcript is isolated and treated with oligo primer and RT to produce mRNA:DNA hybrid
- hybrid is precipitated with cetyltrimetylammonium bromide
- RNA hydrolysis with NaOH
- sense and antisense PCR primers and first round of PCR which produces 2x strand
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Term
| describe the role of linkers in RT-PCR |
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Definition
| Linkers allow cDNA fragents to RE digested and cloned. RE1 and RE2 are different sites so directional cloning is possible |
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| What is the difference between cDNA library, Expression library, and Genomic Library? What type of probes are used for each? |
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Definition
cDNA library: refers to the collection of cloned DNA Expression library: library obtaines when cDNAs are cloned into a bacterial expression vector Genomic library: library obtained when cloning the entire genome of an organism. Use Nucleic Acid probe for cDNA and genomic libraries. Use antibody probe for expression library. |
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| What technique is used in identifying cloned fragments of interest? |
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Definition
Hybridization is used to identify fragments. Stringent hybridization: only one gene forms a stable double stranded complex with probed strands Reduced stringency hybridization: use if trying to identify genes from different organisms, since they'll form stable complexes with the probed strands |
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