PCR

What makes a good primer

1. 3’ end stability

How does ethidium bromide work? Why is is added before the gel electrophoresis

Vir operon

Virulence of Ecoli strain CB1 is largely mediated through expression of the vir operon

Expression of the vir operon is repressed in many CB1 through

Action of VirR protein

VirR protein

• DNA binding protein

• binds to and occules the -10 site of the vir opeon sigma 70 promoter

• contains a ligand-binding domain

• is presence of a putative low-molecular weight inducer, VirR dissocites from the DNA and the transciption of the vir operon proceeds

Bit like lac operon

BUT might be a diffusable furan derivative

Furan?

• Neither E.coli nor humans produce this type of furan

• But non-pthogenic bacteria are knonwn to produce and secrete it

CB1 serovar OX2→ carries wild type gene for virR

• exists as a harmless gut commensal in most hosts

CB1 serovar PaIN

• Carries a small insetion

• disrupt the virR reading

→ In activates VirR protein

→ Causes expression of pathogenicity factors

How identify these genes?

Restirction enzyme sites

• wild type virR→ HindIII restriction fragment, no BamHI cleavage

• PaIN→ disrupt HindIII and induces cleages for BamHI

Steps in practical

• PCR amplifiy the virR gene from various environemtal and clinical isolates

Setting up the PCR

1. PCR master mix

   • VirR 5’primer

   • VirR 3’ primer

   • Taq pol

   • reaction buffer

   • dNTPs

2. add sample

3. PCR:

   • 33 consecutive heating of

   • 30 seconds 94 degrees, 30 at 60 and 1 minute at 72

   • END of EACH SEQUENCE→ hold 5 minutes at 72 degrees (probs to ensure okaski

What is an enzyme unit

• Amount of enzyme required to catalyse conversion of 1 micromol substate per minute

Some add taq at the start, some add it at the end. Why add at the end after reached 94 degrees?

• so do not go through lower temperraeures

• where the primer can bind to not perferctly matching strands

• needs to be at the temperature where the primer will only be on the target strands

• And only then allow to replicate

why are three different temperatures employed during each cycle of the PCR reaction

1. Denature

2. Bind primers

3. Taq polymerase

How do we choose an annealing temperature to use

• 2(A+T) +4(G+C) rule

• or experimentally

can we change annealing temperature of a primer in any way?

• change the 2 +4 rule

• Change its length→ longer = higher temperature

What is the purpose of the long (5 min) terminal extension step at 72 degrees

• Allow for DNA termination

• make sure it is complete

• okaski

Now add restiction enzymes

• Add direcelt to the tube

• THEN:
  - Incubate at 37 degrees for 30 minutes then hold at 4 degrees→ optimal for these specific enzymes

How to set up a gel electrophereis

1. Layer molten agarose along top and bottom of tray

2. sets straight away

3. Place 16 well comb above the cllour strip

4. Add Ethidium Bromide and gently swirl and pour the gel mix into tray

5. remove bubbles

6. Allow to set

7. Pour in the running buffer

8. remove comb

9. run

10. Lift tray out and allow excess EtBr contaminated buffer to run off

What is Ethidium Bromide for?

• Flourcent stain for DNA and RNA

• See under UV light

• inserts itself between base pairs of DNA or RNA

• highly sensitive

• BUT: mutagen and carinogen

How to analyse

1. Analyse under UV light

2. Look at the banding patters and measure distance migrated

3. Compare to the DNA ladder

STrucuture of B DNA

• 1:6 wisth to length?

• golden ratio

• Found strucutre of X-ray diffraction

  • dried out

  • all molecules aligned in acess of fibres

How design primers

1. 18-20 nucleotides

   • not too short (so can stick)

   • not too long (easy to make and not lost too much DNA)

2. Even ATGCs

   • content and distribution

3. 3’ End MUST have a G or C

   • 3’ clamp

   • 3 h bonds→ so stuck down for DNA polymerase

4. Avoid homopolymeric track

   • e.g GGG

5. When need 2→

   • BOTH→ at annealing temp

   • CANNOT be complimentary!!→ will stick together

6. Make sure NOT Shine-dalgarno sequence

Annealing temperautre

2 +4 rule

Ta vs Tm

Ta→ annealing temperautre for primer-template hybridisation

Tm→ melting temperature which is HIGHER than ta

• So 50% of DNA sequence is unbound

ta= tm-2 degrees

Why mad sure reaches the highest possible Ta

• so that cannot bind to anything else

• only the target stuff

• all H bonding is fulfilled

Why only add taq at 93 degrees

• to avoid going through lower temperatures

Alternative

• add beads

• Creates a slurry of wax→ only when the wax melts→ at 93 degress

• does the taq work

Why doesn’t the primer fall off as Temperaure increases?

• Taq still works at lower temperatures

• so binds it and helps raise the melting points

• so primer still stays on

Starting with 1 picogram (10-12 g) of template DNA, and assuming that each round of PCR exponentially amplifies the DNA, how many cycles would be required in order to obtain about 1 µg of product (i.e., enough to yield a visible band on an agarose gel)?

“Exponential growth” describes a growth rate that is dependent on the population size (i.e., a growth curve of the form y = abx ).

The growth rate in a PCR reaction is y = 2n , where y = total number of molecules and n = the cycle number.

2n = 106 (i.e., 1µg/1pg), therefore n(log2) = log(106 ). Thus, n = 6/log2 = ca. 20.

Taq DNA polymerase lacks 3’→5’ exonuclease activity. Consequently, and as might be expected from first principles, Taq exhibits a high error rate (around 3 x 10-4 errors per nucleotide incorporated).

Assuming that we start amplifying from a single copy of the 800 bp virR gene, approximately how many rounds of amplification can occur until it is probable that at least one error has been introduced into the product?

What are the consequences if this error happens to have been introduced at a very early cycle of the PCR? Should Taq be used for cloning purposes?

• Expected errer 1/3×10-4=> 3333

• 3333/800=4.17 fold

• 2^n= 4.17

• n=2.06

??????

In a typical PCR reaction, the annealing temperature is chosen to be just a degree or so below the theoretical melting temperature of the oligonucleotide primer with its complimentary template (why?).

• Tm= 50% at which molecules are associated

• Melting is highly cooperatie

• taking a slightly lower temperature for annealing ensures

  • High specificity

  • most of the primer will be bound to templace

However, once the annealing step is complete, the temperature is rapidly elevated, often to several degrees above the theoretical melting temperature of the oligonucleotide. Why does the primer not dissociate from the template when the temperature is raised this way?

• taq still works at 72 degress C

• resulting longer primer→ has a higher melting temperature

• rate of primer extension at the temp increases from 60 degrees to 72→ must be more than the rate of thermal denaturation of the primer and template!

• PCR would not work other wise

Taq synthesizes DNA at a rate of about 1000 bp per minute at 72ºC. How long would it take for a molecule of Taq to theoretically replicate the entire E. coli genome (4.6 x 106 bp)? Two molecules of the endogenous PolIII enzyme can bidirectionally replicate the E. coli genome in 30 minutes. How many bp per minute can PolIII synthesize? How many turns of B DNA does the PolIII replisome need to unwind each second?

1. 4.6 x 106 /1000 = 4.6 x 103 min = 76.67 hours

1. 4.6 x 106 /2 = bp replicated per replisome = 2.3 x 106 .

2. This is replicated in 30 min (i.e., 30 x 60 sec = 1800 seconds) so each molecule will replicated 2.3 x 106 /1800 bp = 1277bp (=76,667 bp/min).

3. There are ca. 10bp per turn of B DNA, which means that the parent strand will unwind 128 turns per sec (i.e., nearly 7,700 rpm!!!)

Your PCR reactions contained 1 unit (U) of Taq. 1 U of Taq polymerase is defined as the amount of enzyme required to incorporate 10 nMol of dNTP into DNA in 30 minutes under optimal reaction conditions (72ºC). However, your PCR mixtures (25 µL volume) contained 200 µM dNTP. Assuming that these mixtures contained unlimited template DNA, how long would it theoretically take for all of the dNTP present to become incorporated into high molecular weight DNA?

25µL of 200µM dNTP contains (25 x 10-6 ) x (200 x 10-6 ) mol dNTP = 5 x 10-9 mol dNTP (i.e., 5nMol).

Therefore 5nMol/10nMol x 30 min = 15 minutes.

The open reading frame of the wild-type virR gene is 800 bp long. The average molecular weight of an amino acid is 110 Da. The VirR protein is known to be a homodimer. What is the molecular weight of the VirR protein?

1. Each amino acid is encoded by a triplet, so 800bp = 800/3 amino acids = 267 αα.

2. The average Mol Wt of each αα is 110, so the monomer Mol Wt of VirR is 110 x 267 = 29,333.

3. The protein is a dimer, so the dimer Mol Wt = 2 x 29.3 kDa = 58.6 kDa.

1 µg of a particular DNA sample contains 1012 molecules. Assuming that Avogadros Number is 6 x 1023 and that the average weight of a base pair is 650 Da, how many base pairs does the DNA contain?

1. Sample contains 1012/6 x 10^23 = 1.66 x 10-12 Mol in 1 µg.

2. Therefore, 1 Mol of the sample has a Mol Wt of 1/1.66 x 10-12 x (1 x 10-6 ) = 600000 Da.

3. Therefore, if 1 bp has a Mol Wt of 650 Da, the sample contains 600000/650 bp = 923 bp.

Assuming that the average E. coli cell can be approximated as a sphere with a diameter of 1 µm, and that each cell contains an average of 1 genome, calculate the concentration of a gene in the cell.

1. Vol sphere = 4/3 x π x r3 . D = 1 x 10-6 m so r = 0.5 x 10-6 m.

2. Therefore, vol cell (in m3 ) = 4/3 x π x (0.5 x 10-6 ) 3 = 5.22 x 10-19 m3 .

3. There are 1000 liters in a cubic meter so this = 5.22 x 10-16 L.

4. If this volume contains 1 genome (i.e., 1/(6 x 1023) Mol) then the concentration of each genome (or of a single gene, or transcription factor binding site or whatever) in a cell is 3 x 10−9 Mol/L.

Assuming that there is only a single binding site for VirR in the E. coli genome, and that the Kd (dissociation constant) of VirR for its cognate DNA binding site upstream from the vir operon is 3 nM, what fraction of the potential binding sites in the ensemble of cells in a culture will be occupied if the total VirR [dimer] concentration in the cell is 3 nM. Assume that the concentration of DNA in the cell is the same as you calculated in question (viii).

Primers virUP and virDOWN anneal to regions 100bp upstream and downstream of the virR ORF, respectively. The virR5’ and virR3’ primers used in today’s practical anneal precisely to the ends of the virR ORF. A PCR reaction was carried out using the virUP and virDOWN primers and a genomic DNA template.

• When the products were resolved on an agarose gel, a large number of bands were seen, including three closely spaced bands of approximately the expected size (ca. 1 kb).

• The presence of multiple bands is indicative of mis-priming.

• Assuming that you can excise the three ca. 1 kb size bands from the gel, and that the DNA from each can be extracted and purified, outline a strategy that would enable you to determine which of the three bands correspond to the virR amplicon

( “amplicon” is the technical term for an amplified segment of DNA).

• Purify DNA from the three bands and use the resulting DNA as a template for amplification with virR5’ and virR3’…. Because these only attach to the END of the DNA code (so not attach to the start that may look like something else and then bind there etc?)

• Should only get a decent product with the band containing virR.

• This approach is a variant of that family of PCR strategies that come under the aegis of “nested PCR” (look it up in a textbook or even on the WWW)

What strategies might you employ to try and increase the specificity of priming by virUP and virDOWN (see Q(x))?

• Touch-down PCR (look it up!)

  • reduces non-specific priming

  • anneal tempe is gradually reduced

  • annealing temp should be several degrees above the estimated Tm of the pimeres

  • reduce in temperature→ increases specificity of the primer

• try titrating DMSO

• re-design primers to make them longer/anneal to a slightly different site

• or refine the annealing temperature using gradient block PCR.

Prokaroyitc Transcirption

• -35 TTGACA

• -10 TATAAT

• +1 CPuT

Bold is most highly conserved

10% of prokaryotic genes do not have an AUG

• instead have GUG at the start

• coz fMet tRNA => more wobble than other tRNA

How does the Blue white Xgal thing work

• beta galatosidase can hydrolyse a chromogenic XGal

  → Goes blue

• Remain white if no beta galatocidase

DNA binding promoters strucutres→ prokaryotes

1. often are dimers

2. inverted repeats with Dyad symmetry

3. short 4-6 bp

4. Can be half dyad

5. some contain 4 base spacer

6. may not be perfect dyad symmetry

How come enhancer elements can function over distances

• compact tight

• can be flexible