Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction and Purpose

This section aims to provide the general guidelines with a specific protocol, as an example of how to apply nested-PCR to DNA or cDNA obtained from formalin-fixed and paraffin-embedded (FFPE) samples [1–3]. For general requisites for PCR reaction, please refer to the related section in Chap. 20. The goal of this document is to provide a standard workflow for nested amplification, along with a non-isotopical system for PCR product detection. Nested-PCR is related to the reamplification of a first product of PCR by using a second set of primers within the first amplification product. The utilized primers should be different in both reactions. This methodology increases both the sensitivity and specificity of the assay. On the other hand, the risk of contamination increases significantly because of the greater amount of amplification products and working steps involved. In the traditional workflow of nested-PCR, the primers for the second-round PCR were added directly after the first step PCR. Nowadays, only a small amount (a few micro liters) of the first-round PCR product is amplified in a second PCR reaction. Alternatively, to avoid the first-round product contamination, it is also possible to perform a single-tube two-round PCR in single closed reaction tubes [4]. In addition, single-tube two-round PCR could be performed by mixing immediately the primers for the first- and the second-round PCR if they show marked differences in their annealing temperatures. The selected high annealing temperature of the first-round primers results in high specificity in the first-round PCR. This approach has also been described by the use of real-time PCR [5].

The time required for the whole procedure is 3 days.

The extremely high sensitivity of the nested-PCR system could often result in contaminations from previous reactions, then resulting in false-positive analyses. To check the validity of the assay, we strongly recommend using negative controls and taking wide routine precautions. Positive controls are also recommended in the nested-PCR, but to avoid contamination, we suggest using highly diluted controls. We recommend making at least two different sample aliquots and storing them in a laboratory room outside the PCR space. Furthermore, reagent preparation and extractions should be performed in a separate flow-laminar cabinet. First and second nested-PCR rounds should be performed in different areas, e.g. in two different laminar flow hoods, which should be distinct from the extraction ones. In these different spaces, we also suggest employing different supplies, pipette tips, eppendorf tubes, and any common reagent such as deionized water and PCR buffer. Finally, the detection process should be carried out in a dedicated laboratory. Operators are encouraged to wear disposable gloves in each PCR space, changing them frequently, especially when moving from one dedicated area to others.

For its high sensitivity, nested-PCR systems are commonly used in the detection and genotyping of pathogenic viruses and bacteria. Hereafter, we are going to describe a system of nested-PCR to detect borrelial DNA, the causative agent for Lyme disease, in DNA from FFPE specimens. The method presented here has been previously described by Wienecke et al. [6].

2 Protocol

2.1 Reagents

Reagents from specific companies are here reported, but reagents of equal quality purchased from other companies may be used.

  • 10× PCR buffer with MgCl 2 Footnote 1 (stock solution): 150 mM MgCl2, 500 mM KCl, 100 mM Tris pH 8.3 at 25°CFootnote 2

  • Commercial 100 mM stock solution of each dNTP (pH 8)

  • dNTPs: Dilute the dNTPs stock solution to prepare a 10-mM solution of each dNTP in sterile water or DEPC-treated water

  • Primers Footnote 3: Oligonucleotide primers and the method here described for Borrelia detection were designed by Wienecke et al. [6]. The outer primer pair (Bb1 and Bb2) flanks a 171-bp fragment, while the inner primer pair (Bb3 and Bb4) gives a 92-bp product. An internal hybridization control (Bb-hyb) is used. Primer sequences are reported in Table 22.1

    Table 22.1 Primer sequences
    Full size table

  • Lyophilized primers should be dissolved in a small volume of distilled water or 10-mM Tris pH 8 to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10–30 pmol/μl to avoid repeated thawing and freezing. Store all primer solutions at – 20°C. Primer quality can be checked on a denaturing polyacrylamide gel: a single band should be seen

  • Taq DNA Polymerase

  • Agarose powder electrophoresis grade

  • Agarose gel loading buffer (6×): 100-mM EDTA pH 8, 0.25% (w/v) Bromo phenol blue (BMP), 50% (v/v) glycerol in H2O

  • Positively charged nylon membrane for nucleic acid transfer (e.g., Hybond-N+, GE Healthcare)

  • 5× TBE (stock solution): 225-mM Tris base, 225-mM Boric acid, 5-mM EDTA pH 8

  • Ethidium Bromide, 10 mg/ml (stock solution) Footnote 4 Stock solution of EtBr should be stored at 4°C in a dark bottle

  • 0.5-M NaOH, 1.5-M NaCl solution

  • 20× SSC: 3-M NaCl, 0.3-M Na3 citrate.2H2O. Adjust to pH 7 with HCl 1 M

  • N-laurylsarcosine (30% stock solution-Fluka)

  • Poly dATP

  • 10% SDS (w/v) (stock solution) Footnote 5

  • DIG Oligonucleotide Tailing Kit, second Gen. (Roche, cat. No. 03 353 583 910)

  • DIG Nucleic Acid Detection Kit (Roche, cat. No. 11 175 041 910)

  • 3-MM Paper

2.2 Equipment

  • Disinfected Footnote 6 adjustable pipettes, range: 2–20 μl, 20–200 μl, 100–1,000 μl

  • Nuclease-free aerosol-resistant pipette tips

  • 1.5-, 0.5- and thin-walled 0.2-ml tubes Footnote 7 (autoclaved)

  • Centrifuge suitable for centrifugation of 1.5-ml tubes at 13,200 or 14,000 rpm

  • Laminar Flow

  • Cabinet equipped with a UV lamp

  • Thermal cycler: a large number of programmable thermal cyclers marketed by different companies are available for use in PCR. The choice among the commercial instruments depends on the investigator’s inclination, budget, etc. The main requisite is that the instrument must have the licence for use in PCR. For diagnostic purpose, the thermal cycler must be equipped with a heated lid in order to prevent contamination.

  • Horizontal electrophoresis devices

  • UV transilluminator

  • Hybridization oven

  • UV Crosslinker

2.3 Method

  • In a sterile 0.2-ml tube, mix 10 μl of extracted DNA and 40 μl of PCR reaction mixture for a final ­volume of 50 μl. Each reaction contains 10-mM Tris-HCl pH 8.3, 50-mM KCl, 1.5-mM MgCl2, 200-μM each dNTP, one unit of Taq DNA Polymerase, 25 pmol each of the outer primers Bb1 and Bb2. For every PCR run, include a diluted positive control, a bystander DNA that does not contain the sequence of interest (DNA of another bacterial pathogen), a blank control (H2O).Footnote 8

  • Displace the tubes in the thermal cycler. After the initial denaturation for 5 min at 95°C, run 30 amplification cycles as follows: 95°C/1 min; 50°C/1 min ; and 72°C/1 min. Program the instrument in order to maintain the amplified samples at 4°C before storing.

  • Samples and controls derived from the first PCR run are submitted to the nested step.Footnote 9 In a sterile 0.2-ml tube, mix 2 μl of first-round PCR product and 23-μl of PCR reaction mixture for a final volume of 25 μl. Each reaction contains 10-mM Tris-HCl pH 8.3, 50-mM KCl, 1.5-mM MgCl2, 200-μM each dNTPs, one unit of Taq DNA Polymerase, 25 pmol each of the inner primers Bb3 and Bb4.

  • Displace the tubes in the thermal cycler and amplify the target DNA using the previous PCR programme. Set the instrument in order to maintain the amplified samples at 4°C before storing.

  • Analyze 10 μl of the final products by nondenaturing 2.5% agarose gel electrophoresis. In a 100-ml pirex bottle, weigh 1.25 g of agarose powder (electrophoresis grade). Add 50 ml of 1× TBE buffer. Melt the powder in the microwave oven, avoiding loss of material. Cool the gel solution at about 50°C, and add 3 μl of EtBr stock solution. Pour the melted agarose solution into the gel chamber, and let the gel thicken.

  • Load 10 μl of amplification product using 2 μl of loading buffer 6×. Separate the PCR products at 75 V in 1× TBE buffer until the dye reaches ¾ of the gel length.

  • Check the gel at the UV light.

  • Denature the separated PCR products by soaking the gel for two times in the 0.5-M NaOH, 1.5-M NaCl solution for 15 min at room temperature with gently swirling.

  • Prepare the following sandwich for Southern transfer (bottom-up): three sheets of paper 3 MM presoaked in 0.5-M NaOH and 1.5-M NaCl, gel upside down, positively charged membrane over the gel surface avoiding bubble formation,Footnote 10 one sheet of paper 3 MM wet with deionized water, three dry sheets of paper 3 MM, some paper towels, finally a glass with a weight on the top. Allow the DNA to transfer onto the membrane for at least 6 h.Footnote 11

  • Wash the membrane in 2× SSC for 5′, put the membrane between two sheets of paper to dry it, and then store it at room temperature until it is used for hybridization.

  • Before hybridization, crosslink blotted nucleic acids to the membrane at the UV crosslinker twice with face up (each crosslinking takes about 1′ and 18² and goes from 0.25 to 0 J).

  • Perform probe hybridization in 5× SSC, 0.1%-N-laurylsarcosine, 0.02%-SDS, 5-μg/ml poly-dATP, 10-ng/ml of detection oligonucleotide Bb-Hyb, which has been tailed with digoxigenin-dUTP, according to the protocol of the manufacturer (Roche, Dig Oligonucleotide Tailing Kit). Incubate overnight in a hybridization oven at 41°C, swirling gently.

  • Wash the membrane three times with 0.1× SSC, 0.1%-SDS, at 28°C. Detect the system according to the DIG nucleic acid detection kit that uses an enzyme immunoassay and enzyme-catalyzed colour reaction with NBT/BCIP, contained in the DIG Nucleic Acid Detection Kit (Roche).

2.4 Troubleshooting

  • If the bands of the desired product are sharp but faint both in positive controls and samples, inefficient priming or inefficient extension may have occurred. To solve this problem, set up a series of PCRs containing different concentrations of the two primers. Find the optimal primer concentration and then set up a series of PCRs containing different concentrations of Mg2+ to determine the optimal concentration (see Chap. 20, Sect. 20.2.4).

  • If there are bands in the negative controls, contamination of solutions or plastic ware with template DNA may have occurred. In this case, make up new reagents and operate in separate spaces to run PCR and to analyze the products of amplification.

  • If distinct bands of unexpected molecular weight are present, they are the result of nonspecific priming by one or both primers. To solve this problem, decrease the annealing time and/or increase the annealing temperature.

  • If a generalized smear of amplified DNA is present, too much template DNA was introduced.

  • If the amplification is weak or non-detectable, there could be different possible causes: defective reagents, defective thermal cycler, or programming errors. If you are setting up the PCR system, the cause could also be a suboptimal extension of annealed primers, an ineffective denaturation, or the stretches of DNA you have chosen to amplify may be too long.

  • For troubles in hybridization detection, refer to the DIG nucleic acid detection kit data sheet instructions (http://www.roche-applied-science.com/pack-insert/1175041a.pdf).