Modified reaction tubes for the sequential addition of reagents in PCR assays

The polymerase chain reaction (PCR) is used widely for the in vitro amplification of DNA. The versatility of PCR can be enhanced by the introduction of sequential steps such as nested primer amplifications (1) or re-amplification protocols (2). PCR can also be done on cDNA templates produced by reverse transcription of RNA (3). These protocols require additional opening and closing of the reaction tubes with an accompanying risk of sample contamination; an ever present problem with PCR. One way of limiting the number of times a reaction tube is opened is to sequester the additional reagents required for the sequential reactions in the same tube. They are then mixed into the reaction mixture when required. A novel method of achieving this is to compartmentalise the additional reagents in a small reservoir that is contained within the standard reaction tube. Here we describe a simple physical modification to the commonly used PCR reaction tubes to create such a reservoir. Modified Eppendorf 'safe-lock' reaction tubes (0.75 ml) were constructed as follows: a Gilson p20/p200 pipette tip or equivalent (Elkay Lab System) was cut in two with a scalpel blade 10-12 mm from the tip. The tip of the smaller piece was heated in the flame of a Bunsen burner and pressed onto the centre of the inner surface of the lid of a reaction tube at an angle of 90°. The pressure shortened the tip to 7 8 mm and the attached tip formed a reservoir for a second set of reagents. The modified tubes were used in the PCR detection of Hepatitis C, an RNA virus, in the serum of infected patients. Viral RNA was extracted from 200 /tl of serum using the guanidinium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (4) and was dissolved in 15 /tl of DEPC water with 40 U RNasin (Promega). RNA (5 /tl) was incubated at 95 °C for 5 minutes in the presence of the outer primers (0.2 /tM of each) in PCR buffer. These primers are complementary to the conserved 5' non-coding region of the HCV genome (5). After cooling, this mixture was added to a modified reaction tube containing 200 /tM of each dNTP, 1.25 U Taq DNA polymerase (Boehringer Mannheim), 200 U MMLTV reverse transcriptase (BRL) and 10 U RNasin in a final volume of 50 pi. It was overlaid with 2 drops of liquid paraffin. The reservoir contained 5 /tl of PCR buffer, 4 /tM inner primers (5), 1.25 U Taq DNA polymerase and 0.01 % bromophenol blue. This mix was placed as far as possible within the reservoir using a Gilson C10 tip and Vaseline was used to seal the reservoir. The tube was handled with care to avoid premature release of the mix. Reverse transcription was carried out at 42 °C for 30 minutes followed by inactivation of the reverse transcriptase at 95 °C for 90 seconds. Thirty cycles of PCR were used to amplify the outer PCR product from the cDNA (denaturation 92.5°C for 45 sec, annealing 50°C for 60 sec, extension at 70°C for 90 sec). The closed tube was centrifuged for 10 seconds at 6000 rpm in an Eppendorf centrifuge to mix the contents of the reservoir with those in the tube. The reaction tube was tapped a few times to ensure mixing of the reaction mixes, which turned uniformly blue due to the presence of the bromophenol blue dye. A further 30