Term
| name some key features of cloning vectors |
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Definition
- small size = few restriction sites. this makes it easy to manipulate
- they need to be able to replicate within a living cell
- need to have convenient restriction enzyme recognition sites
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Term
| name the early plasmid vector assembled from natural plasmid that was used in jurassic park |
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Definition
pBR322
- this had 40 unique restriction sites
- 15-20 copies per cell
- 2 antibiotic genes (ampicillin and tetracycline)
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Term
| what are multiple cloning sites also known as? |
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Definition
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Term
| how is plasmid DNA introduced to bacteria? |
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Definition
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Term
| electrostatic charges in bacteria can repel negative (both negative). how are divalent metal cations DMC used to overcome this? |
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Definition
DMCs, usually Ca2+ can nuetralise charges.
cells and plasmid mixed at 0C in a CaCl solution. temp > to 42C for a couple of minutes and then returned to 0C. this process known as heat shock causes DNA to enter the cells. |
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Term
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Definition
| cells are mixed with plasmid DNA and a high voltage pulse is applied. this makes the cell membrane transiently permeable to large molecules. A few cells take up plasmid DNA so a method is needed to select those that do (antibiotic resistance) |
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Term
name some advantages of using bacterial plasmids as cloning vectors
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Definition
simple cloning procedure
antiobiotic selection
blue-white screening
small size
transformation is straight foward |
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Term
| what are the limitations in using bacterial plasmids as cloning vectors? |
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Definition
- transformation efficiency relatively low and decreases with increasing plasmid size
- bacterial plasmid best used for small cDNA
- libraries and routine cloning of fragments 30bp to around 15000 bp
- only a few hundred colonies can be grown and screened on a typical plate
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Term
| why can lysogeny genes be replaced with foreign DNA when using the lamda genome for lytic cycle? |
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Definition
lysogeny genes are not requied for lytic cycle.
there is a strict limit on how much and how little can be packed (very specific) |
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Term
| what are the 2 types of vector associated with a lambda genome? |
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Definition
insertion and replacement vector.
insertion: DNA is inserted within modified replacable region (good for cDNA libraries)
replacement: central stuffer region completely replaced (best for genomic libraries) |
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Term
| name the advantages of cloning in lambda vectors (4) |
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Definition
- you can clone upto 25kb
- infection of E. coli by recombinant phages 1000x more efficient than transformation by plasmids
- each arises from a single recombinant phage - contains large numbers of recombinant phages with identical cloned fragments
- hybridisation methods allow screening of more than 50,000 plaques on a single culture plate.
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Term
| name the disadvantages of cloning in lambda vectors (4) |
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Definition
- there is a strict limit on how much or how little DNA can be packaged
- cannot clone more than 25kb fragments
- cells are destroyed by infection
- large amount of vector DNA (25kb) can complicate analysis
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Term
| which sites are involved in rolling circle replication? |
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Definition
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Term
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Definition
a cosmid is a hybrid between a plasmid containing lambda phage cos sites thus cosmid. ha!
these can contain clone frgaments between 35-45kb |
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Term
| name the advantages of using cosmid vectors. (3) |
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Definition
- you can clone fragments of 35-45kb
- it is useful in genome mapping projects
- advantages of using both plasmid and lambda cloning:
- effiecient introduction into bacteria
- antibiotic selection
- large selection of restriction enzyme sites (multiple cloning sites)
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Term
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Definition
| a long continous DNA molecule containing multiple copies of the same DNA sequence linked in series and seperated by cos sites. They are frequently the result of rolling circle replication. eg. if the genes in a phage DNA are arranged ABC then in a concatemer, the genes would be ABCABCABC and so on. |
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Term
| what are BACs and what are their advanatages and disadvanatages? |
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Definition
they are bacterial artificial chrmosomes.
advanatge - large insert size (300kb)
disadvanatge - low copy number (1-2 per cell) |
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Term
| which artificial chromosome is used extensively in genome projects to clone large sections of eukaryotic DNA? |
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Definition
Yeast Artificial Chromosome (YAC)
they can carry inserts as large as 1000kb so fewer clones are needed to make a complete library |
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Term
respectively name the insert sizes for the following:
plasmid, lambda, cosmid, BAC and YAC
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Definition
plasmid = 15kb
lambda = 25kb
cosmid = 35-45kb
BAC = 300kb
YAC = 1000kb |
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Term
| decribe an expression vector |
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Definition
| An expression vector/construct, is usually a plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a traget cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene eg insulin. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. |
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Term
| what vector would you use to express the sysnthesis of insulin in a target cell? |
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Definition
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