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Genetics and Biotech > lecture 12/13 - recombinant dna tech 1/2 > Flashcards

lecture 12/13 - recombinant dna tech 1/2 Flashcards

1
Q

explain what cloning vectors are

A

DNA molecules that
replicate independently of the chromosome in host cells.

They are usually based on plasmids or phage genomes.

Most are circular but a few are linear.

The copy number may be high or low depending on the vector.

Vectors are mainly used in bacteria or unicellular eukaryotes like yeast

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2
Q

what is transformation

A

introduction of recombnant dna molecules into bacteria cells

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3
Q

what are bacterial plasmids

A

stable extrachromosomal elements

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4
Q

what can clooned genes be used for

A

dna sequencing, overproduction of valuable proteins, constructing transgenic organisms

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5
Q

what is electrophoresis

A

movement of charged molecules in an electric field

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6
Q

what is the basis of molecular cloning

A

if a dna frag is inserted into a plasmid vector then you can get multiple copies of the insert easily by purifying the recombinant plasmid

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7
Q

what type of plasmid is ideal to be used as a vector

A

low molecular weight, high copy number and antibiotic resistance gene

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8
Q

why do vectors need the antibiotic resistant gene

A

so that only cells that contain the plasmid can grow on agar
containing the antibiotic

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9
Q

what is a gene library

A

a set of recombinant DNA molecules that represents the entire genome of an organism

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10
Q

what are resistriction enzymes

A

endonucleases that fragment specific sequences of DNA

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11
Q

what type of ends do restrictive enzymes give

A

can be blunt or have staggered cuts with short stretches of single stranded dna (this is cohesive bc the ends can join)

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12
Q

where do restriction enzymes occur and why

A

naturally in bacteria so they can defend themselves against phages

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13
Q

how do bacteria protect their own dna against restriction enzymes

A

by having modification enzymes methylate sensitive sites

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14
Q

what is the point of dna ligase in terms of resitriction enzymes

A

can covalently join ends that were cut by restriction enzymes (but substrates must have the 5’ phosphate and 3’ hydroxyl group)

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15
Q

what dna ligase is used in gene cloning typically

A

T4 ligase

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16
Q

how is vector self-ligation minimized

A

by alkaline phosphatase dephosphorylating the linearized vector dna. this will make it so the vector can only ligate to insert fragments that have 5’ phosphates

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17
Q

what does polynucleotide kinase do

A

catalyzes transfer of y phosphate of ATP to a 5′
hydroxyl group of a linear DNA molecule

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18
Q

what is blue-white selection used for

A

used as a way to determine if there are inserts in the clones (bc they will be white instead of blue)

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19
Q

what are the steps to construct a gene library

A
  1. isolate genomic dna
  2. cut with restriction enzymes
  3. ligrate fragments to vector
  4. propagate recombinant molecules in host organism
  5. select clones containing a required gene
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20
Q

how do you isolate genomic dna from bacteria

A
  1. use non-violent method to disrupt cells
  2. add in highly purified rnase to break down rna
  3. add proteases to degrease cellular proteins
  4. add strong detergents to solubilize membrane lipids
  5. now just purify dna by phenol extraction or ion-exchange chromatography and alcohol precipitation
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21
Q

how are plasmids isolated

A

since plasmids are much smaller than chromosomal dna then treatments like alkaline lysis or boiling will irreversibly denature the chromosal dna but the plasmid will either not denature or renature easily

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22
Q

how many clones are needed to represent an entire genome

A

depends on the size of the genome and the size of insert in each clone (larger the insert means that fewer clones are required)

23
Q

how is genomic dna digested for library construction

A

partially by a restriction enzyme like Sau3A (which cuts GATC)

24
Q

how is partial digestion achieved

A

using very small amounts of enzyme or restricting digestion time

25
why is high molecular weight dna only partially digested
so that only some of Sau3A sites are cut and you can get large fragments
26
how are restriction enzymes inactivated
heating to 68C or by phenol extraction
27
what happens to the partial dna fragments in library construction
they are ligated to a BamHI cut vector and introduced into host strain
28
whats the formula for calculating the number of clones required
N = ln (1 - P) / ln (1-F) N --> number of clones P --> probability of having given sequence in library F --> fraction of genome in each clone
29
what are plasmid vectors used for
cloning fragments of <10 kb bc larger plasmids cannot be transformed into cells efficiently
30
what are phages used for
inject recombinant dna molecules of ~50 kb into bacteria `
31
how has lamda bacteriophage been adapted as cloning vector
central region can be replaced by 20 kb of foreign dna
32
what are cosmids libraries
combine the advantages of phage and plasmid vectors can accomodate 40kb inserts and can be packages into lambda phage particles
33
what does the cosmid vector have
antibiotic resistance gene, plasmid origin of rep and sequences required for packing recombinant dna molecules into lambda phage pasrticles
34
how do cosmids behave as both phage and plasmid
they efficiently inject into recombinant dna into host cells but it replicates as a plasmid
35
how is electroporation helpful for introducing dna molecules
can efficiently introduce very large dna molecules into cells
36
what are artificial chromosomes
vectors that can clone very large fragments (30kb - 100kb OR bigger)
37
what are YACs and what do they have and how big can fragments by
yeast artificial chromosomes that are linear so they have telomeres at each end, origin of rep and centromere that allows segregation of artificial chromosome when yeast host cel divides up to 1million bases
38
what are are BACs and how big can the inserts by
bacterial artificial chromosomes that are based of the F' factor plasmid. up to 350 kb
39
what is a cDNA library
represents all the mRNAs synthesized by a cell or tissue type
40
why can't eukaryotic chromosomal genes be translated properly in bacterial host
bc bacteria cannot splice out introns
41
how are cDNA libraries made
purified mRNAs are converted to DNA by reverse transcriptase and then clone cDNA into a vector to obtain library
42
what are the ways to ensure positive clones are in the library
1. hybridization to a DNA fragment or probe that is related to target sequence 2. reaction with antibodies against protein product of target gene 3. detection of a new biological activity conferred by target gene
43
when can hybridization be used
if a dna fragment is available that is identical or closely related to gene that is to be cloned
44
what is a dna probe
a labelled dna frag used to detect related dna molecules by hybridization
45
how are dna probes labelled
DNA polymerase is used to replicate the DNA in vitro so as to incorporate labelled nucleotides. Radiolabelled nucleotides incorporate 32P or 35S atoms and can be detected by autoradiography. DIG-labelled nucleotides have digoxygenin that can be detected with antibody-enzyme conjugates. The DIG group in the DNA probe binds the antibody which is covalently linked to an enzyme like alkaline phosphatase. A colour-generating substrate for the enzyme is used to stain clones or DNA molecules that have bound the probe
46
what are the steps to screening a library by hybridization
1. have library and replica plate 2. transfer to nylon membrane 3. denature DNA with .5M NaOH 4. hybridize with probe 5. identify positive by enzyme staining (for dig labelled probes) or autoradiography (for radioactive probes)
47
why may oligonucleotide probes be used
bc more often than not there is not a DNA probe available to identify positive clones
48
what are used to help design oligonucleotide probes that hybridize to the gene sequence
partial amino acid sequences
49
how do oligonucleotide probes deal with the fact that most AA have more than one codon that code them
probe is mixtude of all possible sequences
50
how are the oligonucleotides probes created
T4 polynucleotide kinase can be used to catalyse transfer of a 32P phosphate from ATP onto the 5′ end of an oligonucleotide to make a radioactive probe. OR oligonucleotides can be synthesised with a covalently linked DIG label
51
how does immunological selection works
1. colonies/plaques transferred to nylon membrane 2. expose proteins by treating with detergent 3. treat membrane with antibody specific for target gene 4. positive closed bind antibody 4. treat with protein a-enzyme conjugate so the positive clones are stained colorfully
52
how does screening for biological activity work
detect for biological activity that the functional gene will do. can add in substrates or whatnot to produce color reaction
53
what is southern hybridization
a technique used to detect DNA fragments related to a probe
54
explain the process of southern hybridization
1. chromosomal dna cut with restriction enzymes 2. fragments separated by electrophoresis 3. fragments denatured 4. fragments transferred to nylon membrane 5. dna probe is hybridized to membrane 6. hybridizing bands are detected

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