Recombinant DNA Tech

Question 2

Describe transformation

Answer 2

Take up of foreign DNA, combined into the cell

Question 3

Griffith’s experiment

Answer 3

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

Question 4

Transduction and specialized transduction ***

Answer 4

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

Question 5

Transduction and phage

Answer 5

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

Question 6

Bacterial conjugation

Answer 6

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

Question 7

DNA library

Answer 7

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

Question 8

Recombinant DNA definition

Answer 8

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

Question 9

Three goals of Recombinant DNA technology

Answer 9

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

Question 10

Tools of recombinant DNA

Answer 10

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

Question 11

Define clone

Answer 11

Exact copy of a piece of DNA, cell or animal

Question 12

Attenuated vaccine

Answer 12

Live virus with decreased virulence

Question 13

Retrovirus

Answer 13

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

Question 14

cDNA

Answer 14

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

Question 15

Problems with expressing eukaryotic DNA in bacteria

Answer 15

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

Question 16

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

Answer 16

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)

Question 17

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

Answer 17

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

Question 18

Vector

Answer 18

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

Question 19

Useful properties of a vector

Answer 19

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

Question 20

Process for producing a vector

Answer 20

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

Question 21

Gene library

Answer 21

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

Question 22

Advantage of a phage

Answer 22

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

Question 23

Advantage of cDNA library

Answer 23

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.

Question 24

Preparing genomic library

Answer 24

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)

Question 25

Importance of CFU

Answer 25

Screen for the colony that has the entire gene you want

Question 26

PFU

Answer 26

Plaque forming units (for phage library)

Question 27

PCR process

Answer 27

Denaturation (94˚C), Priming (65˚C), Extension (72˚C). Magnifies certain region of DNA.

Question 28

Probing bacterial colonies

Answer 28

Put paper on top, add radioprobe, see where DNA of interest is

Question 30

Southern blot process and uaes

Answer 30

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

Question 31

Why do we use colony blot to detect required clone initially?

Answer 31

Find bacteria with DNA of interest, then digest and find the region of interest

Question 32

Use of microarray

Answer 32

Monitoring of gene expression, Diagnosis of infection, Mutations

Question 33

Microarray description

Answer 33

Contains single stranded DNA on glass or silicon chips; probe with DNA being tested which will bind complimentary, probe fluoresces when it binds

Question 34

Genetic mapping

Answer 34

Locating genes in the genome

Question 35

Restriction fragmentation

Answer 35

Cutting up chromosome etc. with a couple restriction enzymes, then matching where the cut sites are

Question 36

Applications of Recombinant DNA technology

Answer 36

Protein synthesis, Vaccines, Genetic screening, DNA fingerprinting, Gene therapy, Xenotransplants

Question 37

How we insert DNA into cell

Answer 37

Transformation: plasmid; Transduction: phage; (conjugation not used)

Question 38

How did Pasteur develop vaccine against rabies?

Answer 38

Injected aged culture of cholera - most of bacteria dead - didn’t cause disease, became immune

Question 39

Agricultural applications of recombinant DNA

Answer 39

Herbicide resistance, Salt tolerance, Freeze resistance, Pest resistance, Improvements in nutritional value and yield

Question 40

Gene therapy

Answer 40

Remove cells (which have defective genes), insert normal genes, reintroduce cells into patient

Question 41

Subunit Vaccines

Answer 41

Part of pathogen in vaccine; example: capsule

Question 42

3 steps of vaccine making

Answer 42

isolate, inactivate, inject

Question 43

Conjugate vaccine

Answer 43

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

Question 44

Toxoid

Answer 44

toxin that has been denatured, included in vaccines

Question 45

VLP

Answer 45

viral like particles - have antigens

Question 46

Two responses to vaccine

Answer 46

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