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Genetics and Evolutionary Biology pt2 > Amplification of Recombinant DNA > Flashcards

Amplification of Recombinant DNA Flashcards

1
Q

What is a Vector in Genetics?

A
  • An agent (as a plasmid or virus) that contains or carries modified genetic material (as recombinant DNA) and can be used to introduce exogenous genes into the genome of an organism
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2
Q

Bacteria are the production houses of recombinant DNA molecules because…?

A
  • There are a number of systems that allow extra chromosomal replication of DNA molecules, such as cloning vectors.
  • Bacteria are cheap and easy to grow in the laboratory.
  • The exponential nature of bacterial growth means they can easily be grown in very large quantities (e.g. 10^12-10^15 individual bacteria.)
  • Most cloning vectors are either based on plasmid systems or viral chromosomes.
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3
Q

What are some of the essential features of cloning vectors?

A
  • “Origin of replication”, to direct self-replication
  • “Dominant selectable marker”, usually confers drug resistance on the host cell
  • At least one unique “restriction endonuclease cleavage site” (eg. only one EcoRI site in a plasmid)
  • A cluster of unique restriction sites -> aka Polycloning site or multiple cloning site
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4
Q

What are selectable markers?

A
  • Selectable marker genes are conditionally dominant genes that confer an ability to grow in the presence of applied selective agents that are normally toxic to plant cells or inhibitory to plant growth, such as antibiotics and herbicides
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5
Q

What are major grooves and minor grooves?

A
  • The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together. The grooves twist around the molecule on opposite sides.
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6
Q

Where in the DNA do “Dimers” or two of the same RE interact?

A
  • The two restrictive enzymes are on either strand and interact in the major groove
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7
Q

What are Plasmid Vectors?

A
  • Extrachromosomal, double stranded circular molecules of DNA that replicate autonomously
  • Plasmid vectors can readily harbour up to 10 kb of insert DNA
  • Can exist in multiple copies/cell, 2 or 3 to 100’s: called the copy number of the plasmid
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8
Q

Where do Plasmid Vectors come from?

A
  • Naturally occurring in many microorganisms, especially bacteria
  • Some naturally occurring plasmids carry antibiotic resistance genes which are extremely good dominant selectable markers
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9
Q

Why are Plasmids used in experiments a lot?

A
  • Plasmids can easily be purified from bacteria, modified in the laboratory using restriction endonucleases and DNA ligase, and then re-introduced into other bacteria by transformation
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10
Q

Study the diagrams for DNA recombination, the essential features of cloning vectors and Plasmid Vectors

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

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11
Q

What does transformation mean in genetics?

A
  • Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s).
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12
Q

What happens after a plasmid is cut by an RE?

A
  • The DNA fragments from another source of DNA that have been cut by the same RE will hybridise (insert into the plasmid) and then will be sealed into phosphodiester linkages by the enzyme DNA Ligase. Thus creating a recombinant plasmid
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13
Q

What happens once the DNA plasmid has its three components added?

A
  • After the plasmid has had a replication point of origin, a DNA fragment to be cloned and a Dominant selectable marker added then a pool or library of circular recombinant plasmids is thus created
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14
Q

What is special about a pool or library of recombinant plasmids?

A
  • Each Plasmid carries a unique fragment of foreign DNA
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15
Q

What happens after a pool or library of plasmids is created?

A
  • The host cells, for example E.coli bacterial cells, are added to the plasmids, the cells are then treated with calcium chloride to make them permiable to DNA molecules
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16
Q

What happens after the host cells are added to the plasmids?

A
  • Through transformation a few of the host cells take up a recombinant plasmid while most of the other added host cells do not
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17
Q

What is the next step after transformation?

A
  • The bacterial cells are then poured onto a plate of nutrient agar containing ampicillin
  • With ampicillin in the agar, only cells resistant to the drug (cells with the plasmids which contain the dominant selectable markers) are able to grow.
  • At 37 degrees the cells will grow and multiply, because they cannot move on the agar each will produce a separate colony of cells
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18
Q

What happens to the bacterial cells that do not transform?

A
  • The bacterial cells that did not transform and hence take up the resistant plasmid will die
19
Q

How are the plasmids passed on to daughter cells when the cells replicate?

A
  • The replication point of origin allows the plasmid to replicate by using the host cells enzymes
  • Plasmid replication is independent of host cell division however, during cell replication, one of the replicated plasmids are distributed to each daughter cell
20
Q

Eventually how is a clone made from these plasmids?

A
  • As cell replication continues, a colony starts to form and all cells in a given colony contain the same cloned fragment of DNA in their plasmids
  • This means that they will all demonstrate the same genotype and possibly phontype as whatever it was cloned from
21
Q

What is the insert DNA

A
  • An insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism
  • Plasmid vectors can readily harbour up to 10 kb of insert DNA
22
Q

What happens after the transformed bacteria are added to the agar and the ampicillin?

A
  • Transformed bacteria are selected with an antibiotic and the new, recombinant plasmid grown up and purified in large quantity
  • Transformed bacteria then must be screened to see if the plasmid they contain is modified to harbour the DNA of interest, called the insert DNA
23
Q

What are the two major methods of screening?

A
  • Antibiotic sensitivity (eg. pBR322)

- Colour screening (eg. pBluescript)

24
Q

What is Blue-White Screening?

A
  • The lacZ gene encodes b-galactosidase
  • b-galactosidase converts the colorless substrate X-gal (5 Bromo-4-chloro-3-indolyl-a Dgalactopyranoside) into a blue product
  • Cells with b-galactosidase activity produce blue colonies when grown on X-gal
  • Cells lacking b-galactosidase activity produce white colonies.
25
Q

Study the diagram of the process of Blue-White Screening.

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

26
Q

In the Blue-White Screening, which coloured colony will most likely be the one with the required DNA traits?

A
  • Because blue colonies are the bacteria with the intact plasmid and white colonies are the bacteria with the recombinant bacteria, the cells which will possess the required DNA traits will be the white colonies
  • This is because the recombinant plasmids are the ones that have had fragments of DNA added to them
27
Q

Why would you want to take the plasmid out of a bacteria after your experiment?

A
  • The plasmid can be used for other experiments, it can be mutated, recombinated further or added to a different bacteria.
28
Q

What does Lysis mean?

A
  • The disintegration of a cell by rupture of the cell wall or membrane.
29
Q

What is the 1st and 2nd steps

of the Plasmid DNA Minin-prep (Alkaline Lysis Method)?

A
  • Have a Bacteria cell containing genomic DNA and plasmid DNA
  • Add NaOH and SDS which causes cells to lysis
    and plasmid DNA and genomic DNA to denature
30
Q

What is the 3rd step

of the Plasmid DNA Minin-prep (Alkaline Lysis Method)?

A
  • Add acetic acid which neutralises the NaOH. This precipitates out protein, cell debris, and allows plasmid DNA and genomic DNA to renature.
  • However, the large genomic DNA will also precipitate together with protein and cell debris because its renaturation is disordered
31
Q

What is the 4th and 5th steps

of the Plasmid DNA Minin-prep (Alkaline Lysis Method)?

A
  • Following high speed centrifugation, soluble plasmid DNA is in the supernatant and genomic DNA
  • The proteins and cell debris is in the pellet
32
Q

What is the supernatant?

A
  • Denoting the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process.
33
Q

What is the last step of the Plasmid DNA Minin-prep (Alkaline Lysis Method)?

A
  • Plasmid DNA is then precipitated using Isopropyl alcohol or ethanol
34
Q

Study the diagrams of the Plasmid DNA Minin-prep (Alkaline Lysis Method)?

A

-Google doc

35
Q

What are the different types of Vectors other than plasmid?

A
  • There are some vectors that can accept large fragments of DNA. Examples of these are bacteriophage vectors and cosmid vectors
36
Q

What are bacteriophage vectors?

A
  • Bacteriophage is a bacterial viruses
  • Bacteriophage vectors are made from bacteriophage Lambda (λ) chromosome
  • Central one third of λ chromosome (15 kb) is not essential and so can be replaced by foreign DNA
  • The foreign DNA will be transmitted and replicated along with the λ genome
37
Q

What are Cosmid Vectors?

A
  • Cosmid vectors can accommodate much larger DNA inserts, up to 50 kb
  • Cosmids are hybrids between plasmids and λ phage
  • Combine the key advantages on λ phage vectors and plasmid vectors
38
Q

Study the diagram on Alternate Vectors

A
  • Google doc
39
Q

All vectors described so far have been designed for use in E. coli and/or contain the sequences necessary for replication in E. coli, have vectors been created for kingdoms other than bacteria

A
  • In other species different origins of replication and regulatory signals are used
  • Many other vectors have been designed to introduce DNA into yeast, Drosophila, mammals and plants
40
Q

What are shuttle vectors?

A

E. coli is the host of choice so some very useful vectors have been designed to allow replication both in E. coli, and in another species, these are called shuttle vectors

41
Q

Give an example of a shuttle vector

A
  • Allow a yeast gene to be isolated, mutagenised in E. coli and then transferred back to yeast to test the effect of the mutant gene
  • Diagram of this example is on the google doc
42
Q

What are YAC’s?

A
  • Yeast Artificial Chromosomes
  • Genetically engineered yeast minichromosomes able to accept foreign DNA inserts of 200-500 kb
  • Were useful for construction of genome projects, including the human genome project
43
Q

What does a yeast artificial chromosome contain?

A
  • Contains a yeast origin of replication, yeast centromere, two yeast telomeres, a selectable marker, and a polycloning site
  • Diagram of the structure of a YAC in the google doc
44
Q

What are BAC’s and PAC’s?

A
  • Bacterial artificial chromosomes (BACs) and Bacteriophage P1 artificial chromosomes (PACs)
  • BACs and PACs accept 150 300 kb inserts and are less complex than YACs
  • BACs and PACs have now replaced YAC vectors for studying large genomes

Genetics and Evolutionary Biology pt2 (7 decks)

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