PCR labelling labeling protocols

PCR L ABELING OF cloned DNA sequences

Reagents

(a) 10x PCR buffer: Use a ‘no Mg 2+ buffer', usually supplied together with Taq DNA polymerase, and containing 100 mM Tris-HCl, pH 8.3 and 500 mM KCl and sometimes detergents

(b) Unlabeled nucleotide mixture: 2 mM dATP, TTP, dCTP and dGTP each in 100 mM Tris-HCl, pH 7.5 (Note 2)

(c) Labeled nucleotide mixture: 1mM digoxigenin-11-dUTP (Roche), biotin 16 (or 11) dUTP (Sigma, Roche), fluorescein-dUTP or rhodamine-dUTP (see Table 1 for choice of labels) in 100 mM Tris-HCl, pH 7.5

(d) Primers: For labeling inserts in M13 and related plasmids with the same multiple cloning site (e.g. pUC18, pUC19 or pBluescript) use the universal M13 forward and reverse sequencing primers: 5'GTA AAA CGA CGG CCA GT 3' and 5'GGA AAC AGC TAT GAC CAT G 3' or variants (Note 1). The stock primer solutions should be diluted to 10 mM

(e) 50mM MgCl 2

(f) Template DNA (Note 3)

(g) Taq DNA polymerase (1-5 unit/ m l) (At September 2004, we use Promega; until April 2004, we used Bioline routinely).

Method

1. Mix the following in a 0.5 ml microcentrifuge tube for PCR applications (Note 4):

Y m l Water

1.5 m l Unlabeled nucleotide mixture

1.5 m l Labeled nucleotide mixture (Note 5)

1.5 m l Primer 1 (Note 1)

1.5 m l Primer 2 (Note 1)

1.5 m l MgCl 2 (Note 6)

5 m l 10x PCR buffer

X m l Template DNA

____________________

49.5 m l = Total volume

2. Vortex and centrifuge to bring solution to the bottom of the tube, add 0.5 m l of Taq DNA polymerase to each reaction tube. Overlay with mineral oil (e.g. Sigma M3516), two drops per tube (or use a PCR machine with heated lid). Mix again by sharply tapping the tube, and centrifuge to bring to bottom of tube and separate phases.

3. Program your thermal cycler as follows (Note 7)

            1.  93 ° C 5 min

2 . 94 ° C 30 seconds }

3.  56 ° C 30 seconds }

4.  72 ° C 90 seconds } return to step 2  -   35 times

5.  72 ° C 5 min

6.  Hold 15 ° C (Note 8)

4. Check the PCR product (labeling, size and concentration) by agarose gel electrophoresis (Note 9).

5. Precipitate labeled PCR product with ethanol.

Notes

1. Primers can be synthesized to order or purchased: M13 universal sequencing primers, e.g. Sigma P4165 and P4290 or Stratagene #00303 and #300304. T7 and T3 sequencing primers are also suitable for many cloning vectors depending on the insertion site used for cloning. Many companies offer various primers to cater for more unusual insertion sites or other vector systems. Final primer concentration should be between 0.1 to 1.0 mM in the PCR reaction; 0.3 mM is suggested here. The primer sequences used by the PNACL Sequencing laboratory, University of Leicester are: M13 Forward TGT AAA ACG ACG GCC AGT; M13 Reverse CAG GAA ACA GCT ATG ACC; T7 AAT ACG ACT CAC TAT AGG G; T3 ATT AAC CCT CAC TAA AGG G; pGEX5' GGG CTG GCA AGC CAC GTT TGG TG; pGEX3' CCG GGA GCT GCA TGT GTC AGA GG

2. Store in 100 m l aliquots for 3 months at –20 ° C. Some protocols recommend higher concentrations of nucleotides. Even pure nucleotides degrade over 12 months at –20 ° C and should be replaced regularly. dNTP sets are available from e.g. Amersham, Roche, Sigma.

We use dNTP set of BIOLINE (cat. no. is BIO-39025 - four deoxy nucleotides in separate tube of 25 microM quantity- although the other cat. no.'s are BIO-39026 and BIO-39027 of 100microM and 500microM respectively). The concentration of stock nucleotides is 100mM. To make the dNTP mix (10mM each) is dATP= 1 ul + 9ul Molecular biology grade (e.g. from Sigma) water = 10 ul; dGTP= 1 ul + 9ul sigma water = 10ul; dCTP= 1 ul + 9 ul water = 10ul; TTP= 1 ul + 9 ul sigma water = 10 ul. Mix all 4 dNTPs together to give 40ul of 10mM dNTP mix solution.

3. Clean uncut plasmid DNA from standard mini-preparations is diluted 1/50 to 1/1000 in water for use in the reaction. Typically, this amounts to 10 to 30 ng of template DNA (although a wide range, 10 pg to 100 ng, may be used).

4. Many factors affect the results of PCR reactions: amount of template DNA, primer design, enzyme, conditions of amplification and magnesium ion concentration. No amplification or amplification of incorrect template sequences can occur if any is wrong. As the protocol given here describes the amplification of DNA sequence cloned in a plasmid where M13 primers are defined and the annealing at 55°C avoids polymerase pausing at unusual secondary structures of the insert DNA, optimization with multiple reactions and many different controls are not needed. The amount of template DNA is often difficult to estimate and it is advisable to set up test amplification of the desired size of DNA fragment without labeled nucleotide (if necessary adjust the amount of DNA template and other conditions) before repeating with the label in the reaction.

5. Concentration of labeled nucleotide can be important as some haptens may reduce the activity of the enzyme. Possibly, use slightly more labeled nucleotide, especially with direct fluorophores. Biotin, being the smallest molecule of the widely used haptens, is normally accepted best by the enzyme.

6. MgCl 2 concentration may need to be adjusted up or down; normally, a final concentration of 1 to 5 mM is suitable, being 0.5 to 2.5 mM above that of the total dNTP concentration.

7. The M13 primers perform well at a high annealing temperature (55°C), and normally yield large amounts of a single PCR product. Other primers may need different annealing temperatures and more optimization.

8. Many protocols use 4C hold temperature; this keeps the PCR block cooling running continuously and is not necessary as the enzymes are almost inactive at 4C and the reaction mixture is essentially sterile after the 95C steps.

9. PCR amplification and labeling must be tested by gel electrophoresis for amount of amplification, product size and label incorporation (Figure 4.1, see Chapter 3 for methods, also see Note 3). Include a marker track with a known amount of DNA and compare the fluorescence brightness by eye after ethidium bromide staining to measure concentration and include a suitable size marker. When amplified by PCR (where probe length is defined by the distance between the primers), labeled probes run more slowly, because of the large labeled nucleotides, than control DNA amplified without label. It is useful to include control amplification without label to see the retardation as a measure of level of incorporation. If a fluorophore direct label has been used, the fluorescence of the labeled DNA fragment, and of the unincorporated nucleotides, will be visible in the appropriate color.