Quiz 1 - Lecture Component Flashcards
How do Fluorescent proteins get colour
The chromophore (molecule that absorbs light and he reflects colour) inside the barrel structure
Different chromophore =
different colour
What gives diff colours in a barrel
the amino acids in it
Chromophores are chemically altered without enzymes so using
oxidation or light or stuff
PCR is a rapid procedure for
in vitro enzymatic amplification of a segment of DNA
applications of PCR include 6
-cloning DNA or cDNA
-in vitro mutagenis / engeenering
- genetic fingerprints of forensic samples
-assays fo infectious agents
- Quantifying rna
-covid testing
Covid pcr testing ha real time
reverse trancription PCR of nasal swabs
RNA extracted would be
revers transcribes to cDNA
DNA pol wil adda nucleotide to the
3’ OH of the primer, therfore pol needs a primer with 3’oh
You need a primer for
each strand
The primers need to be tightly bound to
the template at 3’ end but u can have some slack on the 5’ end
in the case that dna is denatures, in a new repli the forward primer anneals to
the lower strand and the reverse primer anneals to the upper strand.
PCR Steps 6
- Initial melting at 94 C for ~5 min
- Denaturation of the DNA strands (94-95 C)
- Annealing of oligonucleotide primers (55-65 C)
- Extension/polymerization with polymerase (72 C)
- Repeat steps 2-4 for 25-30 cycles
- Final extension at 72 C for 5-10 min
Desires amplified products appear during the 3rd cycle and the product accumulate
exponentially
equation?
Taq is a
thermostable dna-dependent dna pol and thought to be stable cuz of hydrophobicity at the core of the enzyme and stabilization of electrostatic forces
Taq has 5’ -> 3’’ pol activy but also
5’-> 3’ exonuclease activty but ni 3’ -5’ exo so no proofreading
taqs substrate is
ssdna, primer with 3’oh
fidelity if the
error rate per nucleotide
3’-5’ exo provides
proofreading by removing 3’ terminal mismatches
Primers determine the specificity of amplification and are designes to
minimize amplification of unwanted sequences
Primers in length are 18-24 nt and pairs do not differ by over
3 nt
Primer dont gave a
secondary structure (so like hairpin loops)
No primer
primer interaction at 3’ end???
Magnesium ions are important too
cuz al thermostable pols require free divalent cations for activity, usually bg++ (dntps and primer bing mg++)
Nucleotides like datp, dctp, dgtp and dttp sre important and in high [ ]
are limitjng probs cu of sequestering mg ++
The template dna can be
ss or ds, liner or circular
Denaturations is determined by
tm cuz if temp not high enough AT will denature but CG wont
In annealing if tempo too high no annealing and if too low
non-specific amplification can occur, usually tm05 used to ensure specificty
What is tm
The melting temperature (Tm) is the temperature at which 50% of the double-stranded DNA is changed to single-stranded DNA.
Extension is at or near
optimum temp for pol
The number of cyckes depns on
initial copy number, efficieny of amplification
Why do we need primers for PCR
Cuz they need a hydroxy end so the 3’ end
SOE is
splicing by overlapping
- Site specific mutagenesis
- Creation of chimeric molecules
- Assembly of large segments of DNA
- For making deletions
- For introducing insertions
Explain PCR
you need primers ABCD, b and c put the actual mutation for both directions and AD are for growing out both ends (flank), they can aslo be used for recognition sites for enzymes,
Primers always become
part of the product of PCR
The worst place to put a primer is
the 3’ end but the middle of 5’ end is fine
If you add all the primers together you’ll just get
all the AD together and bc togethr cuz theyre complimentary and it wont work
I dotn get it continue later
review slides
so the melting temp of primers is determined by
their size and GC content
lets say we have a primer with a high tm but the revers primes tm is low, what can we do?
Design a longer primer, cuz the longer the higher the temp
BC primers are how long?
18-35 nucelotides
They also provided adequate
overlap for efficient splicing
Flanking primers, AD are also just so imprtant cuz they can
be used to introduce recognition sites for enzymes
Its not good to have enzyme cut sites
too close to the ends , why?
The length og these primers is
18-35 but at least 10 nts
Deletions work by creating
hairpin loops, so its not transcribed and therefore then not replicatd when it goes up exponentially
The forward primer will begin
at the 5’ end of DNA a
Recombinant DNA is
any DNA molecule made up of sequences/fragements from diff sources
DNA cloning is
preparing large numbers of identical dna molecules
DNA clong is made possible by
using recomb dna methods
You can clone DNA to study genes , structurs, functions and how they are regulated, to do this
you need to isolate the gene and put it into a vehicle or vector to make more recomb DNA? then tranfer thsi to a hos vell which eill replicate with the recomb DNA, WITH THIS IT AMPLIFIES
bACTERIAL dna IS
methylated at certain site and these are charctersitic of th species
This addition of methyl sites
protected the sites from sesive attacks by restricition enzymes
SLIDE 16, 17, 18
Methylation patterns are
maintianed during dna repli
foreign dna that is unmethylated or has diff methylation pattern than host cell
will be degraded
Types I and III are
not very usefull cuz they cleave DNA at sites away from the recognition site and have random cleavage patterns
Type II however
recognizes specific sites and cleaves those sites
Nomeclature
1st letter of genus name, 1st 2 letters of species name, strain types, nth enzymes of that types
Type II restriciton enzymes recoignize
symmetric DNA sequences 4-8 bp
Theyre usually pallindromic
so weather it reads 5’ to 3’ or 3’ to 5’ its the same
They can create either
either sticky ends or blunt ends
Hindiii makes
sticky ends
Smal makes
blunt ends
Ecoti makes
sticky ends
BamHI makes
sticky ends
Practice slide 25
Ligase catalyzes the
formation of a phosphodiester bond between the 5’ and 3’ group
What does it require as a cofactor
ATP
Why is dna ligase needed
cuz when restriction enzymes cut they maker hydrogen bonds to make bade paiors but arent covalently bonded ?
Chimeric restrictions sites
?
DNA vectors can
replicate autonomously in bacteria
They contain unique…
restriction endo nuclease cleavage sites that are present only once in the vector
What do they have to distinguish host cell that has the vector vs those that do
a selectable marker like antibiotic resistance genes
They have to be easy to seperate from the
bacterial chromosomal DNA
Compare the Basis, size limits and Use of Plasmids
naturally occuring;
Under 10 KB;
Subcloning and downstream manipulation, cDNA cloning and expression assays;
Compare the Basis, size limits and Use of Phages
Bactiriophage lambda
5-20kb
Genomic DNA cloning, cDNA cloning and expression libraries
Compare the Basis, size limits and Use of cosmid
Plasmid containing bacteriophage lambda cos site
35-45kb
genomic library contruction
Compare the Basis, size limits and Use of BAC
Escherichia coli f FACTOR PLASMID
75-300 KB
Analysis of large genomes
Explain plasmids
their naturally occurring extra chromosomal double- stranded circular DNA that carry an origin of repli
One of the first plasmids developped for cloning was
pBR322
It was modified to have
an antibiotic resistance gene and multiple cloning sites so basically you can linearize and add insert
What is a very important thing u need to put these inserts in a vector
U need a ligase to seal it shut
Then you transfer the ligation reaction to a
bacterial host and thats where it multiplies
Why use antibiotics
Cus the successful ones with the plasmid will have the antibiotic gene and survive while the ones that didn’t take up the plasmid with die
How does ligation work in this
forms phosphodiester bonds between the 5’ phosphate and the 3’ hydroxy
So why isn’t ligation 100% effective?
cuz the to ends can easily go back together
How can we fix the problem of ends sticking back together without the vector?
Using a phosphatase so removing 5’ phosphate so that is can join back together and have the insert DNA provide a 5’ phosphate.
A second way to avoid ends going back together is to
Cut with 2 different restriction enzymes to make non complimentary sticky ends
So now, u only have the option of having the same restriction enzyme cut, what the issue here
that it could be put in either direction (cuz palindromic?)
How do we make sure thats not happening
with gel electrophoresis, so basically if it cuts in the write spots you’ll get certain bands and if it cuts in the wrong direction you’ll get different one (cuz you put a certainrestriction enzyme within you insert toom tocheck maybe?)
So only transformed bacteria will survive but how do you know which one has recombinant DNA (the insert)?
blue-whote screening so ones with the vector will be white while others will be blue (without)
So technically this works cuz of lac z , explain
so the e-coli has the n-terminal of lac z, and the plasmid will have the N-terminal so when they are together they work and have functional b-galactosidase enzyme, if there’s cleavage of x-gal - blue so it has plasmid, if there is white it doesn’t have plasmid cuz it didn’t cleave anything
restriction enzyme have specific bufferes and they differ by
salt, mg, pH and buffer type
If doing directional cloning it is best to
have enzymes that work together in the same buffer
How does the buffer chart work
u want to use a buffer that’ll work on both so nearly 100%, if you cant find one like that you’d have to do it sequentially so using one enzymes buffer and then the others
How can u use PCR to add in restriction sites
slide 8
Taq has terminal transferase activity so
that adds a single adenosine to the 3’ end of the ThPCR amplified fragment
The 2 types of topoisomerase are
types 1 which cuts 1 strand and type 2 which cuts both strands
TA cloning uses
types 1 topo
The 3 main components of ta cloning are
3’ a overhang on both ends of the taq amplified PCR
vector with 3’ T overhangs
t4 DNA ligase
However, pols that have 3’ - 5’ exonuclease activity or proofreading wont do this so
these products would have to be blunt ended ligated into vectors or As added with taq pol after pcr
Topo q bind to duplex DNA at specific sites and
cleaves the phosphodiester backbone after 5’CCCTT in a strand
The energy from the broken bond is conserved and does what
its conserved by the fromartion of a covalent bond between 3’ phosphate and the cleaved steandf and tyr-274 on topo 1.
The phospho-tyrosyl bnd can be attacked by the 5’ oh
of the og cleaved stran to reverse the reaction and release the topo 1 enzyme
What does TA cliing not require
a ligase
