Recombinant DNA Tech Flashcards

2
Q

Describe transformation

A

Take up of foreign DNA, combined into the cell

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3
Q

Griffith’s experiment

A

R and S strain of streptococcus,, R avirulent, S had capsule and was virulent; combining dead S and live R produced death

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4
Q

Transduction and specialized transduction ***

A

Phage has brought part of previous host into new host. Specialized: only certain sequences transferred

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5
Q

Transduction and phage

A

Phage enters, enters lysogenic cycle, when it enters lytic cycle, takes up DNA from host it, infects next host

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6
Q

Bacterial conjugation

A

pilus, F+; extends and pulls other cell in, can transfer entire chromosome = huge change

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7
Q

DNA library

A

All DNA/genes of one bacteria represented (by using another bacteria with different pieces of the first in plasmids)

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8
Q

Recombinant DNA definition

A

Unnatural DNA made of two or more sequences that wouldn’t otherwise combine; intentionally made using natural and artificial processes

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9
Q

Three goals of Recombinant DNA technology

A

Eliminate undesirable phenotyes, combine beneficial traits, microorganisms that produce things that we need.

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10
Q

Tools of recombinant DNA

A

Mutagens, restriction enzymes, vectors, gene libraries, synthetic nucleic acids

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11
Q

Define clone

A

Exact copy of a piece of DNA, cell or animal

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12
Q

Attenuated vaccine

A

Live virus with decreased virulence

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13
Q

Retrovirus

A

Forms DNA from RNA genome, then incorporates itself into the genome.

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14
Q

cDNA

A

Make double strand DNA from mRNA using Reverse transcriptase, we get just the gene (no introns, easier to isolate mRNA)

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15
Q

Problems with expressing eukaryotic DNA in bacteria

A

Possibly digest DNA (we can mutate that tho). Post-translation modification not accomplished in bacteria. Introns. Reverse transcriptase is error prone

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16
Q

How does reverse transcriptase cause problems with respect to vaccines? What is a possible solution?

A

It makes many errors, which essentially mutate the genome, which can result in changes to antigens so they cannot be targeted anymore. Solution would be to target regions that are absolutely essential therefore could not exist with mutations (ex. adhesins)

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17
Q

Restriction enzymes. What factor in the DNA accounts for the frequency of cutting?

A

Cut pallindromic sequences. Cut blunt or sticky ends. Blunt are more difficult to use but non specific. Frequency affected by GC content.

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18
Q

Vector

A

Plasmid that we cut then insert DNA, used to insert a gene into a cell. Also viral genome or transposon

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19
Q

Useful properties of a vector

A

Small enough to manipulate in a lab, Survive inside cells (replicate), Contain recognizable genetic marker (antibiotic resistance), Ensure genetic expression of gene

20
Q

Process for producing a vector

A

Cut gene and plasmid with restriction enzymes, combine them with ligase, introduce vector into bacteria, plate bacteria to select

21
Q

Gene library

A

Collection of all the genes of a single chromosome (but not always) so you don’t have to isolate them yourself

22
Q

Advantage of a phage

A

Insert more efficiently, and we can clone toxic genes (which would kill bacteria)

23
Q

Advantage of cDNA library

A

DNA made from mRNA. Introns removed and we see only the genes that are expressed (ie in infectious diseases). Doesn’t work for genomic library.

24
Q

Preparing genomic library

A

Isolate DNA (plasmid and genomic DNA), Digest with restriction enzyme, Verify digests (agarose gel electrophoresis), ligate together, Transform into host cell, Screen (antibodies or DNA probe)

25
Q

Importance of CFU

A

Screen for the colony that has the entire gene you want

26
PFU
Plaque forming units (for phage library)
27
PCR process
Denaturation (94˚C), Priming (65˚C), Extension (72˚C). Magnifies certain region of DNA.
28
Probing bacterial colonies
Put paper on top, add radioprobe, see where DNA of interest is
30
Southern blot process and uaes
Transfer DNA from gel to a membrane and probe for DNA of interest; used to differentiate different samples of DNA. Used for forensics, diagnose disease, detect organism that can’t be cultured
31
Why do we use colony blot to detect required clone initially?
Find bacteria with DNA of interest, then digest and find the region of interest
32
Use of microarray
Monitoring of gene expression, Diagnosis of infection, Mutations
33
Microarray description
Contains single stranded DNA on glass or silicon chips; probe with DNA being tested which will bind complimentary, probe fluoresces when it binds
34
Genetic mapping
Locating genes in the genome
35
Restriction fragmentation
Cutting up chromosome etc. with a couple restriction enzymes, then matching where the cut sites are
36
Applications of Recombinant DNA technology
Protein synthesis, Vaccines, Genetic screening, DNA fingerprinting, Gene therapy, Xenotransplants
37
How we insert DNA into cell
Transformation: plasmid; Transduction: phage; (conjugation not used)
38
How did Pasteur develop vaccine against rabies?
Injected aged culture of cholera - most of bacteria dead - didn’t cause disease, became immune
39
Agricultural applications of recombinant DNA
Herbicide resistance, Salt tolerance, Freeze resistance, Pest resistance, Improvements in nutritional value and yield
40
Gene therapy
Remove cells (which have defective genes), insert normal genes, reintroduce cells into patient
41
Subunit Vaccines
Part of pathogen in vaccine; example: capsule
42
3 steps of vaccine making
isolate, inactivate, inject
43
Conjugate vaccine
found an antigen that hasn’t varied for a long time ie Na+ transporter - but too small to make an immune response - so we conjugate 3 copies of the transporter and link it to a flagella gene so it is big enough
44
Toxoid
toxin that has been denatured, included in vaccines
45
VLP
viral like particles - have antigens
46
Two responses to vaccine
antibody response: against antigens in blood; cell mediated: against internal antigens (antigens get inside cell so antibodies can’t access), then cell sends out signal that it’s infected and it gets killed