PL4 Flashcards
describe steps (pathway) of recombinant DNA technology
vector + DNA fragment –> recombinant DNA –> replication of recombinant DNA within host –> isolation, sequencing and manipulation of purified DNA fragment
what is a vector
contents for replicating DNA derived from pieces of DNA that grows in bacteria
what is recombinant DNA
DNA from different sources
bacterial vector with some sort of eukaryotic DNA
want to produce more of that DNA for experiments
what are plasmids
most commonly used vectors
small
multiple copies per bacterial cell - aids replication
describe plasmids - 6
most common vector used in recombinant DNA technologies
circular
dsDNA (double stranded)
extrachromosomal (not in bacterial chromosome - exists separately)
found in bacteria and lower eukaryotes
Replication occurs before cell division (not linked to replication of chromosome, bacterial cells can have many copies of plasmids)
what are restriction endonucleases or restriction enzymes
cleave or cut phosphodiester bones usually in symmetric fashion
Recognize sequence, bind and cut it
where do restriction endonucleases cut
site is symmetric - reads same 5’–>3’ (on both strands) = palindrome
Restriction enzymes recognize palindromes (sites)
what are sticky ends
endonuclease makes staggered cuts at a specific sequence
ECORI = 4 base overhang
these can base pair to each other - only DNA cut with specific enzyme can anneal through sticky end with another piece cut with ECORI
a vector DNA cut with ECORI will only base pair with…
a genomic DNA fragment also cut with ECORI
what does a vector have
replication origin
Restriction enzyme site
some selectable marker
do all endonucleases cut directly at site they recognize
NO
some cut upstream - not useful fro DNA cloning since it will not be compatible for sticky ends
what are blunt ends
some restriction enzymes do not make staggered cut = they cut at same spot on both strands = blunt ends
are blunt ends used in DNA cloning
nO
SInce ligation reaction is not as efficient - since would not be helped by base pairing of sticky ends
maybe sometimes used for a specific reason
describe T4 DNA ligase
broken phosphodiester bonds are fixed by T4 DNA ligase
MAKES new bonds in phosphodiester backbone
seals recombinant DNA molecule
name the parts of engineering E coli plasmid
plasmid cloning vector with replication origin, gene that controls drug resistance (ampr) and polylinker
describe gene that controls drug resistance (engineered E coli plasmid)
want to be able to select bacterial cells that have this plasmid from ones that do not (if they took up drug resistance or not)
petri plating = cells with no plasmid will die on petri plate since plated on drug always
describe polylinker (engineered E coli plasmid)
area with many restriction enzymes
artificial sequence with many restriction enzymes all adjacent to each other - so can use same vector to clone DNA (cut with any of enzymes)
how many restriction sites do we want for vector
1 site in vector
so it will cut then sticky ends have to anneal to fragment of DNA
do not want to cut vector into pieces (bad)
is it useful to use single restriction enzyme or more than one
if use single enzyme then cannot control direction of how fragment inserted into vector - usually fine but if you have specific protein then wrong strand can be transcribed
if use 2 enzymes = cut bacteria with it - 2 ends, then cut vector with same - 2 sites, so each site will match with end of DNA cut by same restriction enzyme = can control orientation of fragment
what is another advantage of using 2 restriction enzymes
sometimes there is noise because vector just ligates back to itself - if done with 2 enzymes then will not happen since sticky ends will be incompatible
what makes up recombinant plasmid
plasmid vector and DNA fragment to be clone - cut with enzyme and produce recombinant plasmid
describe transformation of recombinant plasmid
add DNA to cells and want them to slurp it up
mix E coli with plasmids in presence of CaCl2 and heat pulse = takes up DNA
also can use electroporation = shock so they take up DNA
WHAT happens after transformation of recombinant plasmid
culture on nutrient agar plates with ampicillin = transformed cells survive and cells that do not take up plasmid die on ampicillin plates
plasmid replicates and cell multiplication = colony of cells each containing copies of the same recombinant plasmid
what are DNA libraries
where permanent collections of genes can be obtained and maintained
name and briefly explain 2 types of DNA libraries
genomic libraries = chromosomal DNA
cDNA libraries = represent mRNA present in a given sample
describe how genomic library made
vector cut with BAMHI
4 base sticky end - 6 base recognition site, so 4^6 = BAMHI site comes up every 4096 nts
partially digest with Sau3A = much more frequent since 4 base recognition site (4^4=256) - produces same sticky ends as BAMHI (so compatible)
Sau3A = low concentration and cut small so only cut but not fully
then LIGATE
describe how cDNA library made
uses reverse transcriptase (RT)
RNA –> cDNA (via reverse transcriptase)
RNA –> proteins (via translation)
producing complementary DNA strands from mRNA = complementary DNA (cDNA)
describe generation and cloning of a cDNA copy of mRNA
10 steps
involves oligo-dc primers
make double stranded DNA
linkers for some short sequences that contain EcoRI can be added and gets cut with enzyme and cloned into vectors = have library
what are uses of recombinant DNA construct (2)
where particular gene is expressed
- microarray and in situ hybridization techniques reveal mRNA expression, co regulation and localization
where particular mRNA might accumulate
- recombinant DNA expression vectors enable regulated expression of exogenous genes and production of proteins in prokaryotic and eukaryotic cells
what does in situ hybridization reveal
spatial distribution of an RNA
describe in situ hybridization
make clone of DNA and want to know which cells/tissues in embryo express it
Synthesize a probe that is labelled with biotin and denature probe and so iys single stranded and hybridize so it can get inside tissue
usually use cDNA since whole probe is complementary to mRNA (genomic DNA had introns)
sonic hedgehog mRNA in 10 day mouse embryo = gene tested
only seen in specific places
what does microarray analysis do
Simultaneously measures levels of many mRNAs
allows you to do many hybridization experiments at same time
describe microarray analysis
fibroblasts with and without serum
reverse transcribe to cDNA labeled with fluorescent dye (one colour per type (with/without serum))
mix together and then hybridize to solid support and that is how probes for thousands of different genes
microarray = short oligo peptides, order is known
colours = red (RNA after stimulation), green (RNA before stimulation), yellow (more or less same amount in both)
what can cluster analysis identify
coordinately regulated genes
each column represents a different gene at times after addition of serum
when is lac promoter active
in presence of lactose or molecule like lactose (IPTG)
cloned genes can be expressed…
transiently or stably in cultured animal cells
describe transient transfection
no selectable marker so eventually gets lost on cells
usually good enough for many experiments
describe stable transfection
neomycin resistance
protein gets expressed and then integrated into host chromosome then continues to propagate and cell divides and plasmid replicates
must keep electing on plates with bacteria
what can retroviral vectors be used to do
integrate cloned genes in mammalian genomes
what is first step of using retroviral vectors to integrate cloned genes into mammalian genomes
construct virus that can infect certain cells of organism and integrate the DNA it carries into chromosomes of cells
by transient transfection of cultured cells with 3 different plasmids
describe the 3 plasmids of using retroviral vectors to integrate cloned genes into mammalian genomes
vector plasmid = had 2 sequences derived from transposable elements (LTR - allows it to integrate into chromosome), must be with gene you want to express
packaging plasmid = carries many genes from virus that are needed to make structural components of virus
viral coat plasmid = encodes membrane proteins on surface of virus which determines type of cell virus can infect
what happens when have all 3 components (using retroviral vectors to integrate cloned genes into mammalian genomes)
those with all 3 have all components to assemble virus inside them
so then virus is functional and it infects those cells and comes out of cells - so then virus can be purified
what is a common virus used in using retroviral vectors to integrate cloned genes into mammalian genomes
lentivirus
powerful technology
