Amplicon Analysis

Gel electrophoresis followed by hybridization with labeled DNA probes has been used to verify specific amplification products. If primer pairs are chosen so amplicons differ in size by 50-100 bp, agarose gel electrophoresis can be used to resolve M-PCR amplicons. Figure 1 shows the separation and identification of a 390-bp amplicon for N. gonorrhoeae and a 241-bp amplicon of C. trachomatis produced by M-PCR. With a well-characterized M-PCR assay that gives few or no nonspecific amplification products, gel electropho-

Figure 1 Assessment of the sensitivity of an M-PCR assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA. Serial 10-fold dilutions of C. trachomatis and N. gonorrhoeae DNA were tested by PCR using KL1/KL2 and H01/H03 primers, as described in the text. Amplification products were assessed by agarose gel electrophoresis. Outer lanes contain 1-kb marker DNA. Lane 1:1 pg; Lane 2:100 fg; Lane 3: 10 fg; Lane 4:1 fg; Lane 5: 0.1 fg. Locations of 390-bp and 241-bp amplicons of N. gonorrhoeae and C. trachomatis are indicated by arrows.

Figure 1 Assessment of the sensitivity of an M-PCR assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA. Serial 10-fold dilutions of C. trachomatis and N. gonorrhoeae DNA were tested by PCR using KL1/KL2 and H01/H03 primers, as described in the text. Amplification products were assessed by agarose gel electrophoresis. Outer lanes contain 1-kb marker DNA. Lane 1:1 pg; Lane 2:100 fg; Lane 3: 10 fg; Lane 4:1 fg; Lane 5: 0.1 fg. Locations of 390-bp and 241-bp amplicons of N. gonorrhoeae and C. trachomatis are indicated by arrows.

resis alone is usually sufficient for detecting the presence or absence of specific sequences. During the development of the assay, hybridization with labeled probes should be included to verify specific amplification products. Southern blotting followed by hybridization is necessary for reactions that give many nonspecific products when the specific amplicon is obscured. Some PCR assays may never yield "clean reactions" with easily identifiable amplicons, making hybridization mandatory. Solid-phase hybridization using 96-well microtiter plates coated with avidin to capture biotin-containing amplicons, or complementary capture probes, has been used by many researchers and represents an easy way to assay PCR products for specific sequences. High-performance liquid chromatography (HPLC) has been used to detect specific amplicons and may be well suited to M-PCR for the detection of several amplicons differing in size by <50 bp. Simple M-PCRs with only two or three amplicons can be analyzed with oligonucleotide probes labeled with different enzymes and appropriate substrates giving absorbence at different wavelengths. The availability of dNTPs labeled with fluorescein iso-thiocyanate (FITC), rhodamine, or other fluorochromes presents the possibility of using fluorescence spectroscopy to detect several different amplicons in a single reading of a 96-well plate. Flow cytometry capable of distinguishing and quantifying several different fluorescent labels may also be useful for analyzing M-PCR products although, to our knowledge, this has not yet been reported. Chemiluminescence and other technologies will surely follow, adding to an ever-expanding list of possibilities for detection.

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