Variations on a Theme

In addition to the standard PCR/LDR technique outlined in Subheading 3.2. above, there are several variations of LDR as an assay tool for mutation identification, SNP detection and DNA methylation analysis.

To genotype hundreds of thousands SNPs accurately in multiple samples in a high-throughput format, one variation is to perform LDR on the DNA samples followed by PCR amplification of the ligation products. By performing LDR directly on the DNA samples using primers bearing universal sequences on the 5' end (see Fig. 3), ligation products can be subsequently simultaneously amplified using universal primers. There are several advantages to performing LDR prior to PCR amplification: (1) it eliminates the time-consuming steps of design and optimization of multiplex gene-specific PCR primers; (2) it reduces the cost of synthesizing hundreds of fluorescently labeled allele-specific LDR primers, since product labeling can be accomplished using fluorescently labeled universal PCR primers; (3) it reduces the complexity of the assay system to ensure an accurate and efficient DNA analysis by avoiding the common pitfalls associated with multiplex PCR amplification (e.g., formation of primer dimers and other nonspecific amplicons that may interfere with downstream applications); (4) it reduces the time required for LDR primer design and reaction optimization; and (5) it provides an initial linear amplification of the targeted genomic information that is nonbiased, it promotes allelic balance, and it may minimize the need for later PCR cycles that may detract from this balance. LDR/PCR/Universal Microarray

The use of multiplex LDR followed by PCR was initially developed to score chromosomal instability in tumors (see Subheading 1.4. above). A schematic diagram of multiplex LDR/PCR/Universal Array to determine DNA copy number or score SNPs is shown in Fig. 3. In this approach, the universal primer sequences are added to the 5' end of discriminating LDR primers and to the 3' end of common LDR primers. After ligase detection reaction, the excess unli-gated LDR primers and DNA templates can be digested using 5'^3' and 3'^5' exonucleases. The ligation products are protected from digestion, since blocking groups are added at both their 5' and 3' ends. This exonuclease digestion step reduces the potential of nonspecific hybridization and false-positive results on the universal array readout. The ligation products are simultaneously amplified with universal PCR primers. Only one of the universal PCR primers is fluo-rescently labeled to serve as the detection signal when these amplicons are captured on a universal array. LDR/PCR/Universal Display

ABI recently extended the LDR/PCR concept with the development of an ultra-high-throughput genotyping method, SNPlex. This technology utilizes multiplexed oligonucleotide ligation assay on genomic DNA. Each LDR primer pair was synthesized with universal primer sequences flanking the locus-specific sequences. A unique zip-code sequence is designed within the LDR primers to uniquely identity each LDR product. The excess LDR primers and genomic DNA are eliminated through enzymatic digestion. Consequently, all LDR products may be amplified in a single PCR step with two universal PCR primers, one of which is biotinylated. Biotinylated amplicons may be rendered single stranded and captured on streptavidin-coated plates. Each single-stranded PCR product may be identified by its unique zip-code sequence through interrogation with a set of universal ZipChute probes. These probes have fluorescence labels, unique complementary zip-code sequences, and ABI mobility modifiers. ZipChute probes can be eluted and electrophoretically separated on an ABI 3730xl DNA analyzer. Figure 4 shows an example of a 60-plex reaction using this approach. This technique has been validated on 3,000 SNPs using 96 genomic DNA samples. Compared with other genotyping platforms, the SNPlex system demonstrates 98.7 and 99.2% concordance with dideoxy sequencing and TaqMan assays, respectively. This variation is an alternative approach to existing genotyping methodologies and has the advantage of a robust detection strategy and low DNA consumption.

3.3.2. Bisulfite/PCR-PCR/LDR/Universal Microarray

One application of LDR/PCR/Universal Array is to study DNA methylation. In particular, this variation focuses on the detection of aberrant promoter methylation occurring at the 5-position of cytosine within the CpG dinu-cleotide. Sodium bisulfite conversion of cytosines to uracils is one of the most commonly used methods to study DNA methylation. 5-Methylcytosines are resistant to conversion, and deamination only occurs on unmethylated cytosines. The modified DNA sequences can then either be amplified and sequenced, or one can perform methylation-specific PCR (MSP) to determine cytosine methylation status. Our bisulfite/PCR-PCR/LDR/Universal Array approach provides a sensitive and accurate high-throughput format that can detect methylation status in virtually any gene sequence of interest.

A multiplex PCR-PCR/LDR assay is shown in Fig. 7; (see Color Plate 14 following p. 18) to illustrate this approach. When possible, the gene-specific PCR primers used for multiplexing are designed to avoid CpG sites present in the promoter sequences. As a further improvement to accommodate situations in which bisulfite-modified bases cannot be avoided, pyrimidine and purine nucleotide

Nested Pcr

Fig. 7. (Color Plate 14 following p. 18) Schematic diagram, illustrating the procedure for high-throughput detection of promoter methylation status with the combination of bisulfite treatment, multiplex PCR, multiplex LDR, and universal array approaches. The different fluorescently labeled (Cy3 and Cy5) LDR products are captured on the same addressable array. PCR, polymerase chain reaction; LDR, ligase detection reaction.

Fig. 7. (Color Plate 14 following p. 18) Schematic diagram, illustrating the procedure for high-throughput detection of promoter methylation status with the combination of bisulfite treatment, multiplex PCR, multiplex LDR, and universal array approaches. The different fluorescently labeled (Cy3 and Cy5) LDR products are captured on the same addressable array. PCR, polymerase chain reaction; LDR, ligase detection reaction.

analogs are incorporated within the PCR primers. These modified bases, designated P and K, show considerable promise as degenerate bases. The pyrimidine derivative P base pairs with either A or G, whereas the purine derivative K base pairs with either C or T; thus the target DNA can be amplified regardless of its methylation status. Multiple promoter regions are amplified in a two-stage nested PCR reaction simultaneously. The first-stage multiplex amplification uses pairs of gene-specific PCR primers with universal sequences attached to their 5' ends. The second-stage amplification uses universal PCR primers to amplify the first-stage PCR products. The final PCR products are usually verified on an agarose gel prior to LDR analysis.

The details of LDR primer design have been described in Subheading 3. and in Note 1. Briefly, three LDR primers are designed for each CpG dinucleotide site. Two discriminating primers are labeled at the 5' end with either Cy3 or Cy5 and at the 3' end with a G or A, respectively. The single common primer for the reaction consists of a 5' phosphate and terminates at the 3' end with a zip-code complement sequence. Degeneracy is also accommodated in the LDR primers by using pyrimidine and purine nucleotide analogs. After the LDR products are captured on a universal array, the methylated cytosine residues are detected by the presence of Cy3 signals; the presence of unmethylated cytosines is revealed by the presence of Cy5 signals.

Typically, 1-2 pg of genomic DNA in a volume of 40 pL is incubated with 0.2 N NaOH at 37°C for 10 min. Then 30 pL freshly made 10 mM hydro-quinone and 520 pL of freshly made 3 M sodium bisulfite, pH 5.0 (Sigma, ACS grade) is added. This mixture is next incubated for 16 h in a DNAthermocycler using alternating cycles of 50°C for 20 min followed by a denaturing step of 85°C for 15 s. The bisulfite-treated DNAs can be desalted using MICROCON centrifugal filter devices (Millipore, Bedford, MA) or, alternatively, cleaned with a Wizard DNA clean-up kit (Promega, Madison, WI). The eluted DNAs are incubated with 1/10 volume of 3 N NaOH at room temperature for 5 min prior to ethanol precipitation. The DNA pellet is then resuspended in 20 pL deionized H2O and stored at 4°C. Bisulfite-modified DNA is stable at 4°C for at least 1 mo.

The current assay is designed to detect the extent of DNA methylation within the promoters of the tumor suppressor genes p15INK4b, p16INK4a, p19ARF, p21CIP, p27KIP, p53, and BRCA1, as well as the imprinted gene small nuclear ribonucleoprotein N (SNRPN). Using the same design parameters, the promoter regions of seven additional genes were chosen to investigate their promoter methylation status in human tumors. These include O6 methyl guanine DNA methyl transferase (MGMT), adenomatous polyposis coli (APC), retinoic acid receptor (RARb), tissue inhibitor metalloproteinase (TIMP-3), death-associated protein kinase (DAPK), E-cadherin (ECAD), glutathione S-transferase (GSTP1), and Ras association domain family 1 (RASSF1). The hemimethylated SNRPN is used as a positive internal control.

As seen in Fig. 8 (see Color Plate 15 following p. 178), to demonstrate that LDR primers are working properly, genomic DNAs of normal lymphocytes with and without in vitro methylation are included in experiments as controls. DNA extracted from colorectal cancer cell lines SW1116 and DLD1 is also used to validate this strategy. All experiments were performed minimally in duplicate to avoid ambiguity. For each promoter region, three CpG sites were chosen to analyze their methylation status. The presence of Cy5 signals indicates efficient amplification during multiplexing PCR steps. The promoter regions will be considered to be hypermethylated only when at least two CpG sites can be detected by Cy3 labeling. In most cases, the universal arrays provide very high capture specificity. The methylated promoters identified in this method may be reconfirmed by either bisulfite sequencing or uniplex PCR/PCR/LDR under more stringent hybridization conditions on a fresh array in a separate experiment.

In contrast to MSP-based methods, the bisulfite/PCR/LDR approach circumvents the issues of incomplete bisulfite conversion (C to U modification is not 100% efficient) and the potential primer extension of unmethylated DNA by extension of a G:U mismatch. The requirement of scoring methylation at three CpG sites per promoter using LDR should help the assay retain its exquisite specificity.

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