Term
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Definition
| Conference on DNA modification risk, scientists put a voluntary moratorium on their own work, and set guidelines based on risk estimates. No incidents yet, one of the biggest rules was physical containment |
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Term
| Central dogma in molecular biology is |
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Definition
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Term
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Definition
| the production of an RNA copy of a DNA strand |
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Term
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Definition
| decoding of an RNA molecule to produce a protein |
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Definition
| Commercialised recombinant DNA, discovered several restriction enzymes, founded genetech and a method for producing insulin from bacteria |
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Definition
| Is an easy bacteriophage to work with |
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Definition
| Isolated nerve growth factor, discovered epidermal growth factor and helped develop our understanding of cancer |
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Definition
| Created the first recombinant DNA particles |
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Term
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Definition
| A unit of heredity composed of nucleic acid, a discrete part of the chromosome that determines a particular characteristic, and describes an open reading frame that will be transcribed together with its trancsriptional control elements, (enhancer, repressor, promotor, terminator, etc |
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Definition
| The region of the gene that will be transcribed and translated |
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Term
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Definition
| are the collection of protein encoding genes together with the interspersed non-coding DNA |
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Term
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Definition
| The amount of DNA and genes does not have any effect on the apparent complexity of creatures |
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Term
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Definition
| autonomously replicating DNA molecules that can be used to carry foreign DNA, based on naturally occurring DNA sequences |
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Term
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Definition
| Plasmid, Phage, cosmid, artificial chromosomes (yeast, P1, Bacterial and human artificial) |
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Term
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Definition
| Circular DNA molecules found in bacteria that are replicated by the hosts machinery independently of the genome, accomplished by ori, ccontain selectable markers |
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Term
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Definition
| from 500 copies a cell, to 1 or 2 |
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Term
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Definition
| first cloning vector, which contained an ori from ColE1, two antibiotic resistance genes and unique restriction enzymes |
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Term
| Second generation of plasmid vectors were |
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Definition
| pUC plasmids, with multipple clonign sites and alpha complementation |
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Term
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Definition
| the lacZ gene encodes beta galctosidase which can cleave a colourless substrate called X-gal, forming galactose and an indoxyl derivate which turns blue. The mutant form of lacZ is only functional if LacZ alpha peptide is present. when DNA is incerted it disrupts the alpha peptide production, meaning recombinant colonies will be white |
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Term
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Definition
| M13 is a bacteriophage which infects E.Coli. Developed because the it packages genomes as single stranded DNA. It infects by attaching to the F pilus of E.coli, and the DNA is injected, where it is converted to double stranded form. It is replicated using rolling circle replication, to form single stranded DNA. New virus particles are released without cell lysis |
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Term
| Rolling circle replication |
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Definition
| Starts with a closed circular piece of double stranded DNA, a specialist enzyme nicks the DNA. DNA polymerase synthesizes DNA off teh free 3' end hydroxyl, which pushes the old strand of the template |
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Term
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Definition
| Contain plasmids containing M13 and plasmid oris, allowing it to replicate like a plasmid under most situations and M13 replication can be induced by adding some proteins and enzymres |
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Term
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Definition
| One of the most common phagemids |
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Term
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Definition
| mediates recombination between DNA sequences, by deleting DNA between loxP sites, and it can be turned on or off by researchers during stages of development |
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Term
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Definition
| The phage DNA becomes integrated into the bacterial genome via homologous recombination between attP and attB sites. The prophage DNA remains integrated until it is induced to enter the lytic pathway |
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Term
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Definition
| Lambda insertion and replacement vectors only 32-52 kb can be packaged into the lambda head, this can be done in vityro, because the middle portion of the lambda genome can be replaced if the lytic life cycle is used up to 23 kb DNA can be inserted in lambda genome They can have circular or linear DNA, are easier and morefficient to make libraries than cosmids, and have a higher yield of recombinants, but cannot have long eukaryotic genes |
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Term
| How are lambda plaques made |
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Definition
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Term
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Definition
| a hybrid between lambda and plasmid, the COS sites are the only thing necessary for lambda DNA packaging. if one can ligate COS sites about 50kb apart then the products can be packaged in vitro. Difficult to make libraries and les efficient than lambda, but long eukaryotic genes can be included in a single fragment |
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Term
| How are cosmid vectors made |
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Definition
| colony hybridisation technique |
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Term
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Definition
| ampkification of specific DNA sequences in vitro. requires two primers complementary to each strand of DNA and a DNA polymerase. |
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Term
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Definition
| 94C to break, 55 to attach primers, 72 for free nucleotides |
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Term
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Definition
| Isolated from bacteriums found in hot springs, so it is thermo-stable, but cannot proofread. half life at 95C only 1.6 hours |
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Term
| Nowadays most polymerases used in PCR come from |
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Definition
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Term
| Primers chosen for PCr must |
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Definition
| be 17-30 nucleotides long, at least 50% GC content, annealing temperatures should be equal to around 2(AT) + 4 (GC), long runs of a single nucleotide should be avoided, as should complementary sequences with a primer or between two primers |
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Term
| Why is it necessary to use a cut blunt end rector with added T residue after using Taq polymerase |
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Definition
| Because Taq polymerase adds an A residue to the 3' end |
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Term
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Definition
| uses a complimentary oligonucleotide to prime DNA synthesis from an RNA molecule. The single strand is then used as atemplate for synthesis of a second DNA strand |
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Term
| PCR to detect a point mutation |
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Definition
| The mutant will be longer or shorter than the WT, primers can be made to only fit the mutant, and WT wont replicate, then gel electrophoresis can seperate them |
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Term
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Definition
| highly repetitive sequences of DNA that are essential for proper control of chromosome distribution during cell division |
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Term
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Definition
| consist of DNA and protein, located at the ends of chromosomes protecting them from damage |
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Term
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Definition
| autonomously replicating sequences which contain a yeast centromere and telomere, genes for yac selection, a supressor tRNA gene, bacterial ori and selectable marker, are linear, one copy per cell, unlimited cloning capacity, up to 40% chimerism and unstable` |
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Term
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Definition
| method of finding, isolating and cloning an allele in a gene library |
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Term
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Definition
| illustrate the arrangment of genes and DNA markers |
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Term
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Definition
| randomly breaking up DNA sequences into small pieces and then rassembnling them by looking ofr region of overlap |
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Term
| The suppressor for tRNA in YACS |
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Definition
| expresses tRNA tyrosine, in certain mutant cells, adenine biosynthesis is blocked. This causes an intermediate to accumulate that gives yeast cells a red colour. The expression of the suppressor supresses the mutation and results in the formation fo white colonies. When genomic DNA is inserted, it destroys the suppressor in the EcoRI site, making the colonies red |
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Term
| What is the issue with cloning large fragments in YACS |
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Definition
| They are unstable, prone to breakage and recombination |
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Term
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Definition
| P1 artificial chromosome, lytic pathway can be repressed, maintained as a large, stable, low copy plasmid, two oris, one to control lytic replication, one for plasmid, pac site is cleaved prior to insertion of phage DNA, sacB is the site that encodes levansucrase which is toxic for E.Coli that grow on sucrose, useful for cloning large genes, chromosome walking, physical mapping and shotgun sequencing |
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Term
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Definition
| Engineered version of F1 plasmids, OrisS, F factor ori maintains bacs at 1-2 copies a cell, f factor genes required for replication and maintenance if copy number, low DNA yield, circular, 1-2 copies a cell, low cloning capacity and stable |
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Term
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Definition
| white blood cells isolated and mixed with agarose, enzymes released to break down membranes, restriction enzymes cut DNA, fragments seperated by gel electrophoresis, markers show size of fragments, large ones are ligased together |
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Term
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Definition
| contain dna copies of mrna and are tissue and developmental stage specific dependant on reverse transcriptase and terminal deoxynucleotide transferase which catalyses the addition of nucleotides to the 3' end without needing a template. |
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Term
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Definition
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Term
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Definition
| Remove all cDNA that is common between two seperate libraries, leaving only cell/tissue specific DNA behind |
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Term
| Subtractive hybridisation |
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Definition
| Have a driver and tester cDNA library, label the cDNA with biotin and is in excess. Two pieces of cDNA with biotin cannot bind, but a labelled piece can bind with an unlabelled pice. So driver cDNA cannot bind to itself, and only driver cDNA with complementary sequences can bind with tester DNA. Only cDNA that is not common from the tester cDNA will not be labelled with biotin. Avidin binds with biotin, so when the cDNA is washed through an avidin column, only cDNA unique to the tester will flow through |
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Term
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Definition
| libraries are screened with anitbodies or protein functions, and protein interaction screening is used to identify proteins tha tphyscially associate |
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Term
| Nucleic acid hybridisation |
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Definition
| petri dish with colonies of bacteria containing recombinant plasmids, use a sheet of nitrocellulose to produce a replica of colonies. lyse and denature dna, incubate with probe for specific DNA and wash. exposing to film should reveal the location of the colony containing the plasmid |
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Term
| Immunoscreening of lambda phage libraries |
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Definition
| cDNA cloned into the lambda zap can be expressin the presence of IPTG under the control of the bacterial lac promoter. Recombinant phages expressing the desired cDNA can be identified with specific antibodies. |
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Term
| Characteristics of normal cells |
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Definition
Anchorage dependant,
density dependant inhibition of proliferation,
finite lifespan
, altered characteristics with in vitro age |
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Term
| Characteristics of immortalised cells |
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Definition
continuously culturable,
with infinite lifespan,
reduced requirement for serum and growth factors,
shorter population doubling time,
loss of anchorage dependance,
genetic instability,
altered growth control |
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Term
| Commonly used immortal cell lines |
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Definition
HeLa
NIH3T3 - mouse fibroblast
P19 - mouse embryonal carcinoma
COS-7 monkey african green kidney
CHO - chinese hamster ovary |
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Term
| Mammalian expression vectors |
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Definition
A promoter
, a ribosomal binding site,
a multiple cloning site,
a transcriptional terminator
, a selectable marker gene |
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Term
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Definition
| used to study gene regulatory elements such as enhancer elements |
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Term
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Definition
| used to direct expression of gene products, initiat mRNA synthesis independently of regulation, used to find sequences that aid transcription |
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Term
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Definition
| used to specify expression to target cells, used to find sequences that aid transcription |
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Term
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Definition
| used to control the on off expression of cloned genes, used to find dequences that iad transcription |
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Term
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Definition
| produces neomycin, selects cells in G418, an aminoglycoside that blocks protein synthesis and is similar to kanamycin |
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Term
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Definition
| hygromycin-b-transferase, selects cells in hygromycin B, an aminocylitol, that inhibits protein syntheis. Aids photosynthesis |
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Term
| Mammalian expression vectors |
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Definition
| A promoter, a ribosomal binding site, a multiple cloning site, a transcriptional terminator, a selectable marker gene |
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Term
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Definition
| puromycin-N-acetyl transferase, selects cells in puromycin, an antibiotic that inhibits protein synthesis |
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Term
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Definition
| bleomycin binding protein, selects cells in bleomycin or zeo, and antibiotic that binds DNA and blocks RNA synthesis |
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Term
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Definition
| xanthine-gunanine phosphobosyltransferase, selects cells in guanine deficient media that contains inhibitors of de novo GMP synthesis and xanthine, the selects for gpt+ cells that can synthesize guanine from xanthine |
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Term
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Definition
| gene transfer into animal cells - first described in 1962 |
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Term
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Definition
Direct (microinjector and gene gun)
Chemical (DNA calcium phosphate co-precipitation (simplest) and liposome mediated
Electroporation
Retroviral and adeno-associated viral |
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Term
| Calcium phosphate precipitation |
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Definition
| Cells are washed in phosphate buffer, DNA is added and precipitated using calcium chloride, DNA precipitates are internalised by endocytosis |
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Term
| Liposome mediated transfection |
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Definition
| A mixture of polycationic lipid and a neutral lipid will result in the formation of a unilamellar liposome vesicles that have a net positive charge due to amine heads. Method of choice. Minute spherical sack of phospholipid molecules enclosing a water molecule |
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Term
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Definition
| Electricity is used to make small pores which reseal themeselves after the current is removed. The pores allow DNA to travel into the cell. This will kill a number of cells |
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Term
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Definition
| Variation of electroporation, delivers DNA straight to nucleus |
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Term
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Definition
LTR=long terminal repeat,
Psi = packaging site,
Gag = capsid proteins,
Pol = reverse transcriptase,
Env = surface and trans-membrane protein, must be replication incompetent, integrate randomly into genome so can cause issues |
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Term
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Definition
| A virus of the parvovirus family with a single stranded DNA genome, around 5kb, integrates at a specific locus in humns, most promising for in vivo gene therapy |
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Term
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Definition
| DNA taken up by cells will be maintained for a short period of time before it is degraded and/or diluted from the cell during division, however the short amount of time is sufficient for study |
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Term
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Definition
| 30-40% electroporation, lipofection, 60-80% (nucleofection) and higher for viral |
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Term
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Definition
| Recombinant vector is maintained as an episomal replicon - a unit of genetic material that can replicate independantly from a single ori, plasmid DNA containing a suitable ori will be replicated in ells provided that the large T antigen is expresssed in trans, high copy number will enhance the expression |
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Term
| Who identified DNA as the transforming principle, and how? |
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Definition
| Avery, MAcLeod and Mccarty, discovered that heat killed smooth bacteria could transform live rough bacteria, allowing them to kill mice |
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Term
| Who showed DNA was the genetic material and how? |
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Definition
| Hershey and Chase, labelled DNA with radioactive phosphorus, and protein with sulfate, the cells in the next generation were labelled with phosphorus |
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Term
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Definition
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Term
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Definition
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Term
| Who proved semi-conservative replication and how? |
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Definition
| meselson and stahl, grew E. coli in 15N for many generations, allowed one replication in 14N, DNA was 50/50, 14/15, allowed another replication, one 15/14 band, one 14/14 |
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Term
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Definition
| growth phase for transcription, translation and other activities |
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Term
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Definition
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Term
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Definition
| Another growth period, preperations for cell division take place |
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Term
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Definition
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Term
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Definition
| cuts leaving a 5' overhang |
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Term
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Definition
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Term
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Definition
| cuts leaving 3' overhangs |
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Term
| Modern method of isolating plasmid DNA |
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Definition
| Treat bacteria with sodium dodecyl sulphate in the presence of sodium hydroxide to ensure alkaline pH, SDS disrupts cell membrane and denatures proteins, plasmid DNA remains in solution, neutralisatio results in aggregation of chromosomal DNA, protein-SDS complexes and RNA, then the precipitation is removed via centrifugation, plasmnid DNA is further purified using an ion exchange column |
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Term
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Definition
| southern blotting for RNA |
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Term
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Definition
| southern blotting but use antibodies rather than a DNA probe |
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Term
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Definition
| used to identify proteins that bind to particular DNA sequences |
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Term
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Definition
| a fluorescent protein cloned from a jellyfish that provides a green bioluminescent light in response to a calcium dependent enrgy transfer step involving aequorin |
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Term
| Who first discovered that SV40 could integrate into embryonic cells? |
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Definition
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Term
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Definition
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Term
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Definition
| kills cells that dont express neomycin |
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Term
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Definition
| kills cells that express HSVtk |
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Term
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Definition
| Deletes anything between two loxP sites if they are in the same direction, if they are inverted, it inverses the DNA |
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Term
| How can Cre be controlled |
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Definition
| by switching on the Mx or tetO promoters |
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Term
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Definition
| in the absence of tetracycline (tc), the repressor binds to the tetO operator and represses transcription of the genes. If Tc us absent, the repressor cannot bind |
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Term
| What is the reverse tetO system |
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Definition
| Mutant tetR that binds to tetO in the presence of Tc |
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Term
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Definition
| Induced pluripotent stem cell |
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Term
| What is a klenow fragment? |
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Definition
| A fragment left behind after proteolysis splits DNA polymerase I to remove the 5' to 3' activity. The proteolysis is done by subtilisin |
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Term
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Definition
| Like PCR, performed directly on ds DNA, uses only one primer, uses Taq polymerase - modified |
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Term
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Definition
| uses dATP, dTTP, dGTP..eg, if ATp extends primer base is A, TTP is T... etc |
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Term
| Illumina/solexa sequencing |
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Definition
| uses reversible labelled dNTP terminators, nucleotide is detectable from fluorescent tag |
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Term
| How much did a genome sequence cost in 2013 |
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Definition
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Term
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Definition
| minION sequences individual DNA as they pass through nonopores, as each nucleotide passes through the pore it disrupts a current, and different bases produce different disruptions. It reads five nucleotdies at a time, and has difficulties with long runs of the same nucleotide |
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Term
| For DNA to be put through nanopore sequencing |
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Definition
| it must be cut into large fragments and have adaptors joined |
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Term
| Advs of nanopore sequencing |
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Definition
| Lightweight, portable and easy to use, data becomes available as the DNA is sequenced, generates long sequence from individual molecules |
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Term
| Disadvs of nanopore sequencing |
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Definition
| not commercially avaibl e, require preparation, high error rate, analysis tools under development, high cost per bp |
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Term
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Definition
| Mutate gene to test function, check expression levels with expression assays, use immunoblots to check protein level, test effect on other genes |
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Term
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Definition
| allows a specific change to be introduced somewhere in the middle of a PCR product |
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Term
| What is the IMPACT system |
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Definition
| the protein is expressed with a intein tag and a chitin binding domain at the C terminus, the expressed protein can be purified by binding to chitin beads in a purification column, DDT can be added to induce cleavage of the intein, before elution and collection of the protein |
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