Unit 4 Ch.20: Recombinant DNA technology

different applications for recombinant DNA technology

vaccines

diagnostic testing

gene therapy

DNA fingerprinting

agriculture- GMOs

Plasmids

small circular DNA that exists separate from main Chromosome(s)

recombinant DNA: what is it and how is it achieved?

genetic material from multiple sources joined to create unique sequence, achieved through molecular cloning

4 main steps of creating recombinant DNA

isolate DNA segment to be cloned

select cloning vector

join DNA fragments together

clone rDNA into host organism

restriction endonucleases: what is it produced by, what is it used for, and what action does it take?

produced by bacteria, limits bacteriophage infection

recognizes restriction sites and cleaves

what type of restriction endonuclease is most commonly used? what does it do

type II, cuts at recognition site in predictable patterns

restriction endonuclease: restriction sites

often palindromes: sequence is same forwards and backwards

2 different outcomes of cleavage:

blunt end: no complementary end for base pairing

sticky end: 5’ or 3’ overhang

sticky end cleavage pros and cons

allows specificity with attaching in the correct complementary orientation

same specific restriction enzyme site is needed in isolated DNA sequence and plasmid

blunt end cleavage pros and cons

ligate together any fragments without needing complementary base pairs

less efficient, correct orientation not guaranteed

which region from the DNA must the genome be from. for expressed regions we can isolate the ___ from the ___

any

DNA, RNA

reverse transcriptase

primers: oligo(dT) or random hexamers

cDNA: complementary DNA

gDNA: genomic DNA

Creating DNA with rtPCR

Add oligo primer

Add reverse transcriptase to synthesize cDNA strand

Partially digest RNA

DNA poly I synthesizes second DNA strand

Ligaments seals gap

what process is used to make cDNA from mRNA

reverse transcription

cDNA vs gDNA

represents only genes being expressed in cells at the time the library was made

contains all DNA, noncoding and coding, in a genome

what process is used to amplify a target DNA sequence

PCR

PCR process, and what is needed

template, primers, polymerase, nucleotides, thermal cycler

denature DNA, anneal primers, extend primers, three cycles of amplification (each cycle doubles DNA )

mutations for cloning in primer

Elongating and overlapping primer will introduce mutation to new template for next round of PCR to make final DNA sequence

aids in cloning

study disease causing mutations

mutations alter protein activity

vector

allows amplification of DNA fragment

what are the 3 reasons we use modified plasmids as a cloning vector?

relatively small

self replicating

easy to purify

what are some common features of plasmids?

host specific origin of replication

drug resistance

multiple cloning site or unique restriction enzyme sites

expression plasmids inserted DNA downstream of promoter active in model organism (aka final host)

what enzyme is used to bind together DNA fragments

DNA ligase

isolated target DNA and vector pieces placed together

3 methods used for getting rDNA into the host organism (bacteria or yeast)

how many times does it need to be done?

transformation

transfection

transduction

twice

transformation

uptake of exogenous DNA by bacteria cell- stress out the bacteria

transfection and methods

forced introduction into eukaryotic cell

chemical, lipid based, electroporation

transduction

introduction of exogenous genetic material by a virus

how to confirm expression after the plasmid gets put into the organism

drug resistance

lacZ

lacZ

foreign DNA inserted into the gene

bacteria transformed in the plasmid

bacteria with original plasmid produce b-galactosidase, makes blue colonies

recombinant plasma bacteria do not produce it, makes white colonies

bacteria without a plasmid will not grow

what do we need to consider when choosing a final host

study activity of genetic product

introduce advantageous gene into new system

generate large amount of protein for isolation

applications of recombinant DNA sequencing

identify genetic mutations

forensics

genealogy

what is gel electrophoresis

method to visualized DNA RNA and proteins

what are the steps of agarose gel electrophoresis

load samples into the wells

apply electrical current

migration of DNA and RNA through the pores separate by size

stain gel with dye binding to the DNA and RNA

fragments appear as bands

fragments move towards which charge? why? which fragments travel farther and faster?

DNA fragments move towards the positive charge because DNA is negatively charged

short fragments

what do we use to visualize DNA fragments in the agarose gel

Ethidium Bromide

binds to major groove of DNA

what are different ways of visualizing specific targets

ethidium bromide

UV light

Probe

DNA or RNA with a base sequence complementary to a sequence within the gene of interest

there are different types of probes. what kind of labels can we use to visualize the probes?

what are probes used for?

labeled probes are complementary to the sequence of interest, used to locate individual genes/ sequences among DNA fragments separated by electrophoresis

radioactive label, fluorescent label, enzyme label

difference between southern and northern blots and western blots

DNA vs RNA tracking vs protein tracking

how does blotting work

gel placed on paper that binds to molecule, current moves the molecules from inside the gel to the surface of the paper after gel electrophoresis has denatured DNA

what is FISH, and how is it used

fluorescence in situ hybridization

DNA probes determine chromosomal location

DNA probes to visualize a genes expression in a cell

localize protein or DNA sequence

detecting levels of DNA

PCR and agarose gel- semi quantitative

qPCR quantitatively determines the amount of DNA amplified as the reaction proceeds

qPCR 2 ways to get fluorescence signal

Taq-probe: probe with complementarity to your specific target with both a fluorophore and quenching molecule attached

non-specific dsDNA dye to detect PCR products