Applications of Pcrldr and the Universal DNA Microarray

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When our approaches are combined with PCR, they have been successfully applied to the simultaneous multiplex detection of numerous genetic diseases (see Subheading 1.4.1. below). In our own laboratory, the approach has been validated on hundreds of clinical tumor samples during detection of 19 K- ras and 110 p53 gene mutations in non-microdissected tumors, as well as stool, demonstrating the ability to find mutations despite a large quantity of background normal sequence (5,10,12-14). Our approach has the sensitivity to detect 1 in 100 for a p53 mutation in a wild-type sequence, which is impossible to achieve using standard commercial hybridization chips (3,12).

The Universal DNA microarray allows for the detection of (1) dozens to hundreds of polymorphisms in a single-tube multiplex format, (2) small insertions and deletions in repeat sequences, (3) low-level mutations in a background of normal DNA (3,5,6,17,18), and (4) methylation status of gene promoters. In addition, it requires less manipulation of the DNA. Direct hybridization methods require (1) multiple rounds of PCR or PCR/T7 transcription and (2) processing of PCR-amplified products into fragments or rendering them single stranded. In contrast, our approach allows multiplexed PCR in a single reaction (18) but does not require an additional step to convert product into a single-stranded form.

PCR/LDR and the Universal DNA microarray have been successfully employed in studies that required the following capabilities.

1.4.1. Multiplexed Detection of Single-Nucleotide Polymorphisms and Point Mutations

PCR/LDR has been successfully applied to simultaneous multiplex detection of 61 cystic fibrosis alleles (40,41), 6 hyperkalemic periodic paralysis alleles (42), and 20 21-hydroxylase deficiency alleles (17,43). In addition to point mutations, we demonstrated that PCR/LDR could detect instability within the transforming growth factor-^ type II receptor gene and the APCI1307K mononucleotide repeat allele in DNA derived from both blood and paraffin-embedded tumor samples (4,6). The cystic fibrosis test is commercialized by our corporate collaborators, ABI and Celera Diagnostics, and is used throughout the world for prenatal testing of this inherited disease.

1.4.2. Multiplexed PCR for Amplifying Many Regions of Chromosomal DNA Simultaneously

We have developed a coupled multiplex PCR/PCR/LDR assay for use in armed forces personnel. This technique was developed to mitigate the problems of false amplicons, allele dropout, and uneven amplifications, which often mar attempts to perform highly multiplexed PCR (18). A comparison of LDR profiles of several individuals demonstrated the ability of PCR/LDR to distinguish both homozygous and heterozygous genotypes at each locus (18). Others have independently validated the use of PCR/PCR in human identification to amplify 26 loci simultaneously (44) or ligase-based detection to distinguish 32 alleles, although the latter was in individual reactions (45). We have also developed a PCR/PCR/LDR assay to detect the founder Jewish BRCA1 and BRCA2 insertion and deletion mutations associated with breast cancer (8).

1.4.3. Multiplexed LDR/PCR to Determine DNA Copy Number or Score SNPs

We initially developed multiplexed LDR followed by PCR to score chromosomal instability in tumors (2). Others have extended our approach to detection of deletions in the DMD gene, deletions in the hMLHl and hMSH2 genes, and chromosomal trisomy (46-48). Our corporate collaborators at ABI have extended our LDR/PCR protocol for typing single-nucleotide polymorphisms (SNPs) directly on genomic DNA. In their protocol, one of each LDR primer pair contains a unique zip-code sequence, and locus-specific sequences are flanked by universal primer sequences. Consequently, all LDR products may be amplified in a single PCR step, and each product may be identified by its unique zip-code sequence. The products may be rendered single stranded and hybridized on a universal array (Fig. 3; see Color Plate 10 following p. 18), or alternatively used to capture premade fluorescently labeled "zip-chutes" (developed by ABI) (Fig. 4; see Color Plate 11 following p. 18), each with a unique size, for scoring by electrophoretic separation. The technique has been validated on 3000 SNPs using 96 genomic DNA samples (11).

Dna Color Separation

Fig. 3. (Color Plate 9 following p. 18) Schematic diagram for the detection of multiple single-nucleotide polymorphisms (SNPs) using LDR/PCR/Universal Array. Multiplexed LDR is performed on multiple SNPs using allele-specific primers containing unique zip-code sequences and a 5' universal sequence (Un2) as well as locus-specific primers containing a different universal sequence (Un1) on their 3' ends. Only if there is perfect complementarity at the junction will the ligation product form, thus distinguishing different SNPs. Unligated products are destroyed with X exonuclease (5'^3') and exonuclease 1 (3'^5'). All remaining LDR products are coamplified simultaneously with universal PCR primers Un2 and Un1. In this illustration, Un2 is labeled with Cy5 on the 5' end, and Un1 may be phosphorylated on the 5' end, allowing for the option to convert PCR products to a single-stranded form with X exonucle-ase prior to hybridization. PCR products are hybridized to universal array containing zip-codes. Fluorescent signal at a given address scores for the presence or absence of each SNP. LDR, ligase detection reaction; PCR, polymerase chain reaction.

Fig. 3. (Color Plate 9 following p. 18) Schematic diagram for the detection of multiple single-nucleotide polymorphisms (SNPs) using LDR/PCR/Universal Array. Multiplexed LDR is performed on multiple SNPs using allele-specific primers containing unique zip-code sequences and a 5' universal sequence (Un2) as well as locus-specific primers containing a different universal sequence (Un1) on their 3' ends. Only if there is perfect complementarity at the junction will the ligation product form, thus distinguishing different SNPs. Unligated products are destroyed with X exonuclease (5'^3') and exonuclease 1 (3'^5'). All remaining LDR products are coamplified simultaneously with universal PCR primers Un2 and Un1. In this illustration, Un2 is labeled with Cy5 on the 5' end, and Un1 may be phosphorylated on the 5' end, allowing for the option to convert PCR products to a single-stranded form with X exonucle-ase prior to hybridization. PCR products are hybridized to universal array containing zip-codes. Fluorescent signal at a given address scores for the presence or absence of each SNP. LDR, ligase detection reaction; PCR, polymerase chain reaction.

Fig. 4. (Color Plate 11 following p. 18) Electrophoretogram of 30-plex detection in genomic DNA. The data demonstrate the ability of LDR/PCR to characterize 60 alleles simultaneously in an SNP genotyping assay. The blue and green peaks are either FAM or Vic labeled mobility modified zip-chutes. The x-axis is the size of zip-chute and the y-axis is fluorescent intensity of capillary electrophoresis. The zip-chutes have the same color but different motilities for both of allele-1 and allele-2. For example, 1T (first blue peak) is homozygous, T; 17C (green) and 17G (green) are heterozygous C/G, etc.

Fig. 4. (Color Plate 11 following p. 18) Electrophoretogram of 30-plex detection in genomic DNA. The data demonstrate the ability of LDR/PCR to characterize 60 alleles simultaneously in an SNP genotyping assay. The blue and green peaks are either FAM or Vic labeled mobility modified zip-chutes. The x-axis is the size of zip-chute and the y-axis is fluorescent intensity of capillary electrophoresis. The zip-chutes have the same color but different motilities for both of allele-1 and allele-2. For example, 1T (first blue peak) is homozygous, T; 17C (green) and 17G (green) are heterozygous C/G, etc.

1.4.4. Detection of K-ras, BRCA1, BRCA2, and p53 Mutations Using Multiplex PCR/LDR and Zip-Code Capture

Since the zip-code sequences remain constant and their complements can be appended to any set of LDR primers, our zip-code arrays are universal, and we and our cancer collaborators at The Rockefeller University and the Institut Curie have applied this array-based mutation detection to mutations in the K-ras, BRCA1, BRCA2, and p53 genes (3,8,9,12-14). Figure 2 shows the schematic and results for p53 mutation analysis, where by 110 mutations could be queried simultaneously. Mutations present at 1% of the wild-type DNA level, or in pooled samples could be distinguished (8,12).

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