| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Medical Research Council Laboratories, Atlantic Road, PO Box 273, Banjul, The Gambia; fax 220-496513, e-mail skaye{at}mrc.gm
The use of molecular methods such as PCR to quantify nucleic acids has become widely established for both research and diagnostic applications. Increasingly, such methodologies recognize the need for inclusion of internal standards and controls. These are used to define the efficiencies of nucleic acid extraction, reverse transcription, amplification, and detection and to quantify the effects of nonspecific inhibitors. Examples include the "Qs" controls included in Amplicor PCR assays (Roche Molecular Systems Inc.) (1) and the "Qa, Qb, Qc" controls used in Nuclisens assays (Organon Technica) (2). Control constructs (RNA or DNA) are derived from the nucleic acid sequence that the test method is designed to assay and span the primer binding sequences used in the assay. Thus, when added to the test sample, the control is coamplified in the reaction with a single primer pair. To distinguish the control from the test sequence during the detection phase of the assay, a short sequence between the primer binding sites is replaced by a defined sequence to allow differential probing with labeled oligonucleotides.
Nucleic acid controls are usually 1 kb or longer, as shorter lengths may not by efficiently purified by the commonly used silica capture or precipitation methods. Previously, the construction of such controls required the identification or engineering of unique restriction sites between the primer binding sites to allow excision of the wild-type sequence and replacement with the probe sequence (1)(2). The process also required ligation of the probe sequence into the target sequence backbone and cloning into a plasmid containing a transcription promoter in the case of RNA controls. The method described here simplifies the procedure to a two-stage PCR amplification and allows the probe sequence to be placed anywhere between the primer binding sites without the need to identify or engineer restriction sites. Similar approaches have been described previously (3)(4)(5), although none of these is as simple or universally applicable as the method described here. As an example, the method has been used to synthesize a molecular construct for use as an internal standard in a quantitative PCR method for measles virus (MV) RNA.
The principle of the method is shown in Fig. 1
. In step 1, the target gene is PCR-amplified in two sections. The antisense primer (B) used in the PCR of the upstream target sequence and the sense primer (C) of the PCR for the downstream target are sited immediately either side of the desired probing site. These two primers are 5'-tailed with the required probing sequence. After amplification of the two sections, the PCR products are purified to remove the remaining primers (step 2). In step 3, the two amplicons are mixed and reamplified with the two outer primers (A and D) only. Melting of the amplicons from step 1 allows reannealing of the complementary probe sequences. The annealed sequences prime extension to a full-length sequence that can then be PCR-amplified by primers A and D. If the control required is an RNA sequence, a T7-promoter site is included in primer A or D to allow transcription, followed by DNase treatment to remove the template. Finally (step 4), the DNA or RNA construct is purified and quantified by spectrophotometry (absorbance at 260 nm) before dilution and storage at -70 °C for use. The method was used for the construction of an RNA internal standard for use in a quantitative PCR method for the N-gene of MV (3).
|
The first-round PCR products (step 1) were amplified in a single-tube reverse transcription-PCR (RT-PCR) from 
10 000 RNA copies of the Edmonston–Zagreb vaccine strain of MV (6). The RT-PCR reaction volume was 50 µL when the Titan One-Tube PCR reagent set (Roche Biochemicals, Lewes, UK) was used according to the manufacturer’s instructions. Primers were 5'-ACC AAA CAA AGT TGG GTA AGG-3' (bases 1–21 of the MV N-gene) and 5'-GCT CGT ACT CTT TGA TTG AAA TAA GGA TAA TAT ACC TAT T-3' (bases 346–327 plus a randomized tail) to synthesize the upstream portion of the construct. To synthesize the downstream portion, the primers were 5'-TTC AAT CAA AGA GTA CGA GCT TGA TTC AGA GGA TCA CCG A-3' (N-gene bases 367–386 with randomized tail) and 5'-CTG CCC GCG CAA CAT GGA CAT AAT ACG ACT CAC TAT AGG GAG AAC TCA AGT GTG GAT AAC TCA-3' (bases 1000–981 plus T7 promoter). Thermal cycling conditions were as follows: 1 cycle of 50 °C for 30 min; 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 68 °C for 1 min; and 1 cycle 68 °C for 7 min. Amplification was confirmed on a 2% ethidium bromide-stained agarose gel. The products of the two PCRs were pooled, purified on a spin column according to the manufacturer’s instructions (Qiagen), and eluted into a final volume of 50 µL. A 100-µL second-stage PCR (step 3) was used to amplify 2 µL of the purified products of step 1. The reaction mixture contained 2.5 U of AmpliTaq Gold (ABI), 0.2 mM each deoxynucleotide triphosphate (Promega), 0.2 µM each of primers A and D (see Fig. 1
), and 1.5 mM MgCl2 in AmpliTaq Gold reaction buffer. Reaction conditions were as follows: 1 cycle of 95 °C for 8 min; 25 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min; and 1 cycle of 72 °C for 7 min. The products of this second PCR were purified on spin columns (Qiagen) after excision of the specific 1043-bp band from a 2% agarose gel. RNA transcripts were generated in a 100-µL reaction containing 50 U of T7 polymerase (Life Technologies), 0.5 mM each nucleotide triphosphate, 0.01 mM dithiothreitol, and T7 reaction buffer (Life Technologies). The reaction was incubated at 37 °C for 2 h before 10 U of DNase I (Life Technologies) were added. DNA digestion was carried out at room temperature for 15 min and stopped by the addition of 0.2 mM EDTA; the DNase was inactivated by incubating at 65 °C for 10 min. Finally, the RNA transcripts were purified by the method of Boom et al. (7) and eluted into 100 µL of nuclease-free water. After quantification of the final product by spectrophotometry (absorbance at 260 nm), the internal standard was stored at -70 °C in Tris-EDTA buffer containing 1 g/L tRNA (Life Technologies). The performance of the control was assessed in a quantitative RT-PCR based on a previously described method (8). The sense and antisense quantitative PCR primers were targeted to bases 278–299 and 433–413, respectively. The MV-specific probe was complementary to bases 347–366, and the probe for the control sequence was complementary to the randomized sequence inserted into the control construct.
All PCR products generated in the synthesis of the control construct were of the predicted size on an agarose gel. After purification of the final product, the RNA yield was 10 µg in a volume of 100 µL. For a 1000-base sequence, this represented 1011 RNA copies/µL. The control construct was quantified by end-point dilution in the chemiluminescence assay described. The results are shown in Table 1
and are given as ratios of test signals to the mean signal generated by negative (water) controls. Signal ratios generated by wild-type probe reacted with PCR products from the control construct were all close to unity (i.e., there was no cross-reaction), as were those from wild-type PCR products reacted with the control sequence probe. End-point dilution of the control construct confirmed the spectrophotometric estimate of 1011 RNA copies/µL. Assay of the final construct for DNA copies showed that this contained 105 DNA copies/mL (10-6 of the RNA copies). It was intended that the RNA construct be added to the final assay at a concentrations of 100 RNA copies/reaction. At this dilution, the DNA contaminants would be diluted to <0.001 copies/reaction; thus it was decided not to risk the integrity of the RNA construct by the use of more vigorous DNase treatment.
|
Quantitative molecular assays, particularly quantitative PCR, have become an important tool in medical research and routine clinical investigation, particularly for the assay of microbial genomes in clinical samples (9) and for the quantification of gene expression in mammalian cells (10). In such assay systems, accurate quantification depends on standardization and control of all steps in the protocol. In the case of quantitative PCR, this includes controls for nucleic acid extraction efficiency, amplification efficiency, and nonspecific inhibition of the reaction (11). Additionally, there is a need for accurately quantified internal standards with which to compare unknowns to obtain absolute nucleic acid copy numbers in the sample.
The method described here allows molecular constructs for use as internal controls in quantitative molecular assays to be synthesized in a simple two-step PCR protocol. The simplicity of the procedure allows controls to be synthesized in laboratories that do not have facilities available for molecular biology procedures beyond PCR, such as diagnostic laboratories that use molecular detection methods. The method has been applied to the control of a quantitative PCR for MV RNA but could also be applied to the generation of DNA targets. Controls may be synthesized that are appropriate to any source of nucleic acid (e.g., mRNA in the quantification of microbial or mammalian gene expression) that is being detected or quantified by PCR or any other molecular method.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
