Term
| DNA from two sources is isolated and .................. with the same .................. endonuclease |
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Definition
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Term
| the two types of DNA pair at their .............. ends when mixed together. The enzyme DNA ............ joins them |
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Definition
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Term
| the plasmid is inserted into the bacterial cell by ................ |
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Definition
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Term
| Clones are screened for........ of interest |
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Definition
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Term
| which DNA is used for genome mapping and sequencing? |
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Definition
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Term
| which type of DNA contains introns? |
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Definition
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Term
| why is genomic DNA no use for expressing protein in bacteria? |
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Definition
| because of its introns and very long fragments (>20kb fragments) |
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Term
| what category of DNA is chromosomal DNA? |
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Definition
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Term
| what is cDNA synthesised from? Which enzyme is used in this process? |
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Definition
| synthesised from mRNA using reverse transcriptase |
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Term
| apart from reverse transcriptase what 2 other things does cDNA need? |
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Definition
primer
nucleotides:-
dATP dGTP gCTP gTTP
this produces ss cDNA |
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Term
| the second strand of cDNA also requires primer, nucleotides and which enzyme? |
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Definition
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Term
| what is the difference between DNA and cDNA? |
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Definition
| cDNA is intron-less. it is an artificial gene |
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Term
| which enzyme degrades RNA when making cDNA? |
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Definition
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Term
| which enzyme adds nucleotides on the 3" end of ssDNA? |
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Definition
| terminal transferase enzyme |
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Term
| small pieces of DNA that can be used as carriers are also known as? |
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Definition
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Term
| which extrachromosomal bacterial DNA often carries antibiotic resistance? |
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Definition
| Plasmids -these are often used as vectors |
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Term
| in relation to symmetry, what do all restriction enzymes have? |
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Definition
| a 2-fold rotational symmetry |
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Term
| restriction enzymes recognize sequences which maybe how long? |
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Definition
| 4, 6 or 8 nucleotides long |
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Term
| restriction enzymes are isolated from bacteria. what are their roles in protecting the bacteria from invasion? |
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Definition
| they cut up invading DNA protecting the bacteria from infection |
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Term
| what is the correct name for restriction enzymes? |
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Definition
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Term
| what type of DNA will restriction endonucleases cut at defined sites? |
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Definition
Double Stranded DNA
Restriction Endonucleases will not cut ss DNA |
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Term
| if there is a 4 base recognition sequence what is the probability that a restriction endonuclease will cut a piece of DNA? |
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Definition
1/4 x 1/4 x 1/4 x 1/4 = 1/256
on average every 256bp |
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Term
| where on a DNA strand would a restriction enzyme cut? |
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Definition
| the sugar-phosphate backbone |
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Term
| which enzyme joins the sticky ends together to make recombinant DNA? |
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Definition
DNA ligase
DNA ligase joins 5’ phosphate to 3’ hydroxyl in double- stranded DNA
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Term
In a cloning experiment, DNA fragments are joined to a ................
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Definition
vector
these are
Relatively small DNA molecules
a single site is usually for chosen restriction enzyme
capable of replicating in host organism
Contain selectable gene (eg antibiotic resistanc |
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Term
| name 3 hosts that can be used to culture recombinant dna? |
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Definition
bacteria eg. ecoli but not 0157 strain
Yeast
Animals cells in culture |
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Term
true or false?
over 3/4 of the host cells take up a plasmid during transformation? |
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Definition
false
only a fraction of the host cells take up a plasmid during transformation |
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Term
| how can you minimise religation of plasmid vector? (prevent the cleaved sections from rejoining) |
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Definition
treating linearised vector with phosphotase.
this removes phosphate groups and minimises the plasmid from rejoining |
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Term
| name a method used to identify antibiotic resistant cells that also contain recombinant DNA? |
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Definition
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Term
| in blue-white screening, the vector contains a gene that codes for the enzyme B-galactosidase. what is this gene called? |
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Definition
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Term
| what is X-gal and what does B-galactosidase do to X-gal? |
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Definition
| x-gal is a derivative of lactose and this enzyme converts x-gal into a blue colour |
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Term
| in blue-white screening which colour colonies are the recombinant clones? |
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Definition
the white ones - this is because
no enzyme will be produced as the gene is disrupted by insertion of a DNA frgament
no blue product
white colony identified as recombinant clone |
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Term
| what is a library of clones? |
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Definition
where a large number of different genomic fragments or cDNAs are inserted into a vector
you get a mixture of recombinant DNAs each with a different insert. each colony derived from a single recombinant DNA molecule = library of clones |
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Term
| how is the DNA of a bacterial cell protected from its own restriction endonucleases? |
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Definition
| by the addition of methyl groups to adenines or cytosines to the sequences recognised by the enzyme |
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Term
| why is genomic library also known as the shot gun approach? |
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Definition
| no specific gene is targeted for cloning and you will have a mixture of recombinant DNA |
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Term
| what is colony hybridisation? |
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Definition
| where a nucleic acid probe (either DNA or RNA) is synthesised complimentary to the gene of interest. this probe is labelled with an isotope or fluorescent tag so we can track it |
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