Recombinant DNA Tech Flashcards
Describe transformation
Take up of foreign DNA, combined into the cell
Griffith’s experiment
R and S strain of streptococcus,, R avirulent, S had capsule and was virulent; combining dead S and live R produced death
Transduction and specialized transduction ***
Phage has brought part of previous host into new host. Specialized: only certain sequences transferred
Transduction and phage
Phage enters, enters lysogenic cycle, when it enters lytic cycle, takes up DNA from host it, infects next host
Bacterial conjugation
pilus, F+; extends and pulls other cell in, can transfer entire chromosome = huge change
DNA library
All DNA/genes of one bacteria represented (by using another bacteria with different pieces of the first in plasmids)
Recombinant DNA definition
Unnatural DNA made of two or more sequences that wouldn’t otherwise combine; intentionally made using natural and artificial processes
Three goals of Recombinant DNA technology
Eliminate undesirable phenotyes, combine beneficial traits, microorganisms that produce things that we need.
Tools of recombinant DNA
Mutagens, restriction enzymes, vectors, gene libraries, synthetic nucleic acids
Define clone
Exact copy of a piece of DNA, cell or animal
Attenuated vaccine
Live virus with decreased virulence
Retrovirus
Forms DNA from RNA genome, then incorporates itself into the genome.
cDNA
Make double strand DNA from mRNA using Reverse transcriptase, we get just the gene (no introns, easier to isolate mRNA)
Problems with expressing eukaryotic DNA in bacteria
Possibly digest DNA (we can mutate that tho). Post-translation modification not accomplished in bacteria. Introns. Reverse transcriptase is error prone
How does reverse transcriptase cause problems with respect to vaccines? What is a possible solution?
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)
Restriction enzymes. What factor in the DNA accounts for the frequency of cutting?
Cut pallindromic sequences. Cut blunt or sticky ends. Blunt are more difficult to use but non specific. Frequency affected by GC content.
Vector
Plasmid that we cut then insert DNA, used to insert a gene into a cell. Also viral genome or transposon
Useful properties of a vector
Small enough to manipulate in a lab, Survive inside cells (replicate), Contain recognizable genetic marker (antibiotic resistance), Ensure genetic expression of gene
Process for producing a vector
Cut gene and plasmid with restriction enzymes, combine them with ligase, introduce vector into bacteria, plate bacteria to select
Gene library
Collection of all the genes of a single chromosome (but not always) so you don’t have to isolate them yourself
Advantage of a phage
Insert more efficiently, and we can clone toxic genes (which would kill bacteria)
Advantage of cDNA library
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.
Preparing genomic library
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)
Importance of CFU
Screen for the colony that has the entire gene you want
PFU
Plaque forming units (for phage library)
PCR process
Denaturation (94˚C), Priming (65˚C), Extension (72˚C). Magnifies certain region of DNA.
Probing bacterial colonies
Put paper on top, add radioprobe, see where DNA of interest is
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
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
Use of microarray
Monitoring of gene expression, Diagnosis of infection, Mutations
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
Genetic mapping
Locating genes in the genome
Restriction fragmentation
Cutting up chromosome etc. with a couple restriction enzymes, then matching where the cut sites are
Applications of Recombinant DNA technology
Protein synthesis, Vaccines, Genetic screening, DNA fingerprinting, Gene therapy, Xenotransplants
How we insert DNA into cell
Transformation: plasmid; Transduction: phage; (conjugation not used)
How did Pasteur develop vaccine against rabies?
Injected aged culture of cholera - most of bacteria dead - didn’t cause disease, became immune
Agricultural applications of recombinant DNA
Herbicide resistance, Salt tolerance, Freeze resistance, Pest resistance, Improvements in nutritional value and yield
Gene therapy
Remove cells (which have defective genes), insert normal genes, reintroduce cells into patient
Subunit Vaccines
Part of pathogen in vaccine; example: capsule
3 steps of vaccine making
isolate, inactivate, inject
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
Toxoid
toxin that has been denatured, included in vaccines
VLP
viral like particles - have antigens
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
