TaqMan Real TimePolymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N-Acetyltransferase-1 (NAT1) and -2 (NAT2)

N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) exhibit allelic variation and genetic polymorphism associated with increased susceptibility towards drug toxicity and environmental disease (Hein et al., 2000a; Butcher et al., 2002). The variant NAT1 (Table 4.15.1) and NAT2 (Table 4.15.2) alleles possess various combinations of single nucleotide polymorphisms (SNPs), deletions, and/or insertions compared to the reference alleles, that are sometimes inappropriately termed wildtype. Combinations of nucleotide polymorphisms dene alleles. The consensus listing of NAT1 and NAT2 alleles is maintained by an international committee and published at: http://www.louisville.edu/medschool/pharmacology/NAT.html (Hein et al., 2000b).

UNIT 4.15

REAL-TIME TAQMAN ANALYSIS OF NAT1 AND NAT2

TaqMan allelic discrimination methods have been developed to rapidly determine NAT1 (Doll and Hein, 2002) and NAT2 (Doll and Hein, 2001) genotypes. The SNPs selected for NAT1 genotype determinations are: C97 T (R33 Stop), C190 T (R64 W), G445 A (V149 I), C559 T (R187 Stop), G560 A (R187 Q), A752 T (D251 V), T1088 A (3 UTR), and C1095 A (3 UTR). The SNPs selected for NAT2 genotyping determinations are: G191 A (R64 Q), C282 T (silent), T341 C (I114 T), C481 T (silent), G590 A (R197 Q), A803 G (K268 R), and G857 A (G286 E). All NAT2 and NAT1 alleles, except very rare ones, are detected with these SNP determinations. The authors provide detailed methods and materials for using the 7700 Sequence Detector from Applied Biosystems. These methods can be adapted for other instruments from Applied Biosystems or other manufacturers. The specic primers and probes for NAT1 and NAT2 genotype determinations are listed in Tables 4.15.3 and 4.15.4, respectively.

BASIC PROTOCOL

Materials 2 TaqMan Universal PCR Master Mix (Applied Biosystems) 3 M primers (see Tables 4.15.3 and 4.15.4; Applied Biosystems) 4 M uorescent probes: normal, turbo, and MGB (see Tables 4.15.3 and 4.15.4; Applied Biosystems) 5 to 25 ng/l high-quality genomic DNA samples Optical 96-well plates (Applied Biosystems) Optical caps (Applied Biosystems) Sequence detector (e.g., 7700 Sequence Detector, Applied Biosystems) and computer

Techniques for Assessment of Chemical Transformation Contributed by David W. Hein and Mark A. Doll
Current Protocols in Toxicology (2004) 4.15.1-4.15.11 Copyright C 2004 by John Wiley & Sons, Inc.

4.15.1
Supplement 22

Table 4.15.1 Human NAT1 Allelesa

NAT1 allele NAT1 3 NAT1 4 NAT1 5 NAT1 10 NAT1 11A NAT1 11B NAT1 11C NAT1 14A NAT1 14B NAT1 15 NAT1 16 NAT1 17 NAT1 18A NAT1 18B NAT1 19 NAT1 20 NAT1 21 NAT1 22 NAT1 23 NAT1 24 NAT1 25 NAT1 26A NAT1 26B NAT1 27 NAT1 28 NAT1 29

Nucleotide change(s) C1095 A None G350,351 C, G497-499 C, A884 G, T1088 A, C1095 A C344 T, A40 T, G445 A, G459 A, T640 G, 1065-1090, C1095 A C344 T, A40 T, G445 A, G459 A, T640 G, 1065-1090 C344 T, A40 T, G459 A, T640 G, 1065-1090, C1095 A G560 A, T1088 A, C1095 A G C C
560 976

Amino acid change(s) None None ,

1105

R117T, R166T, E167Q None V149I, S214A V149I, S214A S214A R187Q R187Q R187Stop

9 between 9 between

9 between

A
1095

559

T A A T
1088

[AAA] immediately after 1091, C

190

None R64W None None R33Stop None M205V D251V None E261K I263V
1095

3 between 1064-1087, T 3 between 1065-1090 C T T

402 97

A, C

1095

C G T A G A

A A T G A

613 752

777

781 787

[TAA] insertion between 1065 and 1090, C [TAA] insertion between 1065 and 1090 T G, T T
1088 21 777

None None None None None

C A,
1025

[TAATAA]deletion between 1065 and 1090 A, C

1095

a From http://www.louisville.edu/medschool/pharmacology/NAT.html

Prepare reagents 1. Thaw an aliquot of 2 TaqMan Universal PCR Master Mix, primers, and probes at 37 C.
TaqMan Universal PCR Master Mix (Applied Biosystems) is optimized for TaqMan reactions and contains AmpliTaq Gold DNA polymerase, AmpErase, dNTPs with UTP, and a passive reference to correct for well-to-well variation. None of the above reagents are toxic or require special care. All reagents should be aliquoted, to minimize freezing and thawing, and stored at 20 C. Primers and probes for each SNP are listed in Tables 4.15.3 and 4.15.4. The primers and probes are diluted, in sterile DNAse-free water, to a concentration of 3 M and 4 M respectively prior to being aliquoted and stored at 20 C. Fluorescent probes should be kept out of direct sunlight to avoid degradation.

TaqMan-PCR Genotyping of NAT1 and NAT2

4.15.2
Supplement 22 Current Protocols in Toxicology

Table 4.15.2 Human NAT2 Allelesa

NAT2 allele NAT2 4 NAT2 5A NAT2 5B NAT2 5C NAT2 5D NAT2 5E NAT2 5F NAT2 5G NAT2 5H NAT2 5I NAT2 5J NAT2 6A NAT2 6B NAT2 6C NAT2 6D NAT2 6E NAT2 7A NAT2 7B NAT2 10 NAT2 11A NAT2 11B NAT2 12A NAT2 12B NAT2 12C NAT2 12D NAT2 13 NAT2 14A NAT2 14B NAT2 14C NAT2 14D NAT2 14E NAT2 14F NAT2 14G NAT2 17 NAT2 18 NAT2 19

Nucleotide change(s) None T341 C, C481 T T341 C, C481 T, A803 G T341 C, A803 G T341 C T T T T
341 341

Amino acid change(s) None I114T I114T, K268R I114T, K268R I114T I114T, R197Q

C, G C, C T, T C, C T, T A

590

A
759 481

481

T, C

T, A T, A

803 803

G G C G

I114T, K268R I114T, K268R I114T, K268R, I287T I114T, L137F, K268R I114T, R197Q R197Q R197Q

282

341

C, C

341 341

481

T, A T, C

803 481 590

G, T T, A A

859

C, A T, G T, G T, G A T, G A T

411

803

C C C T C C C C C C C

282 282

341

C, G A

590

590

282

590 282

A, A T, G A A

803

R197Q, K268R R197Q R197Q G286E G286E E167K None S287 frameshift K268R K268R K268R D122N, K268R None R64Q

111

C, C

590

481

590

G G

857

282

857

499

481 481

T, 859del G
803 803

803

282 481

T, A T, A T A

G G G

G G G G G G G G A A C

364

A; A

803

282

191 191 191 191 191 191 191 434 845

A, C A, T A, C A, T C C

282

T
481

R64Q T, A A G
803

341

C, C T, G G

R64Q, I114T, K268R R64Q, R197Q R64Q, K268R R64Q, I114T, K268R R64Q, K268R Q145P K282T R64W

282

590

A, A A, C

803

341

C, A T, A

803

282

803

190

a From http://www.louisville.edu/medschool/pharmacology/NAT.html

Techniques for Assessment of Chemical Transformation

4.15.3
Current Protocols in Toxicology Supplement 22

Table 4.15.3 Primers and Fluorogenic Probes for NAT1 SNP Determinationsa, b

Primer or probe 97-Forward primer (58-85) 97-Reverse primer (131-107) [74 bp] 97C-TaqMan MGB probe (90-104) 97T-TaqMan MGB probe (90-105) 190-Forward primer (143-165) 190-Reverse primer (220-198) [78 bp] 190C-TaqMan MGB probe (196-185) 190T-TaqMan MGB probe (195-183) 445-Forward primer (395-416) 445-Reverse primer (506-481) [70 bp] 445G-TaqMan MGB probe (439-452) 445A-TaqMan MGB probe (437-454) 559/560-Forward primer (497-521) 559/560-Reverse primer (595-571) [99 bp] 559C/560G-TaqMan MGB probe (552-566) 559C/560A-TaqMan MGB probe (552-569) 559T/560G-TaqMan MGB probe (552-567) 752-Forward primer (713-734) 752-Reverse primer (793-765) [81 bp] 752A-TaqMan MGB probe (743-762) 752T-TaqMan MGB probe (743-762) 1088/1095-Forward primer (1041-1066) 1088/1095-Reverse primer (1136-1112) [96 bp] 1088T/1095C-TaqMan MGB probe (1105-1079) 1088A/1095A-TaqMan MGB probe (1106-1079) 1088T/1095A-TaqMan MGB probe (1106-1078)

Sequence 5 -gacttggaaacattaactgacattcttc-3 5 -caatggatgttaaggttctcaaagg-3 FAM-ccagatcCgagctgt VIC-ccagatcTgagctgtt 5 -tggacttaggcttagaggccatt-3 5 -gatgattgacctggagacaccat-3 FAM-caccccGatttc VIC-accccAatttctt 5 -ggcagcctctggagttaatttc-3 5 -tactgttcccttctgatttggtctag-3 FAM-ccttgtGtcttccg VIC-tgccttgtAtcttccgtt 5 -gggaacagtacattccaaatgaaga-3 5 -ttgttcgaggcttaagagtaaagga-3 FAM-caaatacCGaaaaat VIC-caaatacCAaaaaatcta TET-caaatacTGaaaaatc 5 -ccctcacccataggagattcaa-3 5 -tttctatttcttcctcactcagagtcttg-3 FAM-acaatacagAtctaatagag VIC-acaatacagTtctaatagag 5 -gaaacataaccacaaaccttttcaaa-3 5 -aaatcaccaatttccaagataacca-3 FAM-atctttaaaaGacatttAttattatta VIC-catctttaaaaTacatttTttattatta TET-catctttaaaaTacatttAttattattat

a Adapted from Doll and Hein, 2002. Nucleotide positions are indicated in parentheses. PCR product size is indicated in square brackets. Nucleotide-specic polymerase chain reaction (PCR) primers and uorogenic probes were designed using Primer Express (version 1.5; Applied Biosystems). The uorogenic MGB probes were labeled with a reporter dye (either FAM or VIC or TET) specic for one of the possible nucleotides identied at eight polymorphisms in the NAT1 coding region or 3 UTR. The uorogenic probes for the 559/560 and the 1088/1095 are distinguished using a three-probe system that has both nucleotide polymorphisms in the same probe. This is necessary because the 559/560 and 1088/1095 are too close together to use the conventional two-probe TaqMan assay. The nucleotide-specic primers amplify a segment of the NAT1 gene anking the probes. b Abbreviations: bp, base-pair; FAM, 6-carboxyuorescein; MGB, minor groove binder; NAT1, N-acetyltransferase 1; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; TET, tetrachloro-6-carboxyuorescein; UTR, untranslated region; VIC, a proprietary dye from Applied Biosystems.

2. Prepare a master solution of 2 TaqMan Universal PCR Master Mix, primers, and probes to have 12 l solution/sample. Make up the solution with the following composition:

TaqMan-PCR Genotyping of NAT1 and NAT2

50% 2 TaqMan Universal PCR Master Mix 10% each appropriate primer 2.5% each of two or three appropriate probes 25% sterile, DNAse-free H2 O.

4.15.4
Supplement 22 Current Protocols in Toxicology

Table 4.15.4 Primers and Fluorogenic Probes for NAT2 SNP Determinationsa

191-Forward primer (131-149) 191-Reverse primer (242-220) [112 bp] 191A-TaqMan probe (204-179) 191G-TaqMan probe (202-181) 282-Forward primer (196-216) 282-Reverse primer (321-299) [126 bp] 282C-TurboTaqMan probe (270-293) 282T-Turbo TaqMan probe (270-294) 341-Forward primer (278-305) 341-Reverse primer (377-359) [100 bp] 341T-TaqMan probe (349-329) 341C-TaqMan probe (349-329) 481-Forward primer (438-458) 481-Reverse primer (549-525) [112 bp] 481C-TaqMan probe (495-468) 481T-TaqMan probe (495-467) 590-Forward primer (483-506) 590-Reverse primer (636-609) [154 bp] 590G-TaqMan probe (577-604) 590A-TaqMan probe (577-606) 803-Forward primer (763-789) 803-Reverse primer (839-819) [77 bp] 803A-Turbo TaqMan probe (816-790) 803G-Turbo TaqMan probe (817-791) 857-Forward primer (768-792) 857-Reverse primer (912-888) [146 bp] 857G-TaqMan probe (842-870) 857A-TaqMan probe (841-869)

5 -gtgggcaagccatggagtt-3 5 -gtggtcagagcccagtacagaag-3 FAM-acaccacccacccTggtttcttctta- TAMRA VIC-accacccacccCggtttcttct-TAMRA 5 -gggtggtgtctccaggtcaat-3 5 -gtgaaccatgccagtgctgtatt-3 FAM-agggtatttttaCatccctccagt-TAMRA VIC-agggtatttttaTatccctccagtt-TAMRA 5 -tttacatccctccagttaacaaatacag-3 5 -ccagacccagcatcgacaa-3 FAM-tgccgtcaAtggtcacctgca-TAMRA VIC-tgccgtcaGtggtcacctgca-TAMRA 5 -gccttgcattttctgcttgac-3 5 -ctttggcaggagatgagaattaaga-3 FAM-cctgatttggtccaGgtaccagattcct-TAMRA VIC-cctgatttggtccaAgtaccagattcctc-TAMRA 5 -ggaccaaatcaggagagagcagta-3 5 -agacgtctgcaggtatgtattcatagac-3 FAM-acgcttgaacctcGaacaattgaagatt-TAMRA VIC- acgcttgaacctcAaacaattgaagatttt-TAMRA 5 -tttaaaactctcactgaggaagaggtt-3 5 -acgagatttctccccaaggaa-3 FAM-cttaaatatatttTtcagcacttcttc-TAMRA VIC-tcttaaatatatttCtcagcacttctt-TAMRA 5 -aactctcactgaggaagaggttgaa-3 5 -tgggtgatacatacacaagggtttat-3 FAM-ccaaacctggtgatgGatcccttactatt-TAMRA VIC-cccaaacctggtgatgAatcccttactat-TAMRA

a Adapted from (Doll and Hein, 2001). Nucleotide positions are indicated in parenthesis. PCR product sizes are indicated

in brackets. SNP-specic polymerase chain reaction (PCR) primers and uorogenic probes were designed using Primer Express (Applied Biosystems). The uorogenic probes are labeled with a reporter dye (either FAM or VIC) and are specic for one of the two possible bases identied at seven SNPs in the NAT2 coding region. b Abbreviations: bp, base-pair; FAM, 6-carboxyuorescein; NAT2, N-acetyltransferase 2; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; TAMRA, 6-carboxytetramethylrhodamine; VIC, a proprietary dye from Applied Biosystems.

Once all the components have been added, mix briey by vortexing.
Aerosol-free tips should be used for all pipeting to minimize risk of cross-contamination.

3. Pipet 10 l of the mix into the appropriate number of wells of an optical 96-well plate. 4. Add 1 l of genomic DNA (5 to 25 ng) to the appropriate wells (one DNA sample/well). Place optical caps on wells.
Make sure not to damage the caps when putting them on the 96-well plate.
Techniques for Assessment of Chemical Transformation

5. Vortex plate to mix the genomic DNA into the mixture. Transfer the 96-well plate to the 7700 sequence detector.

4.15.5
Current Protocols in Toxicology Supplement 22

Prepare the 7700 Sequence Detector 6. Turn on the 7700 Sequence Detector and computer.
Turn on the 7700 Sequence Detector at least 30 min in advance.

7. Open the sequence detector software and close the screen that appears by clicking the upper left-hand corner. 8. Click on File and scroll to New Plate. Select Single Reporter, 7700 Sequence Detector, and Real-time, and click OK. 9. Highlight the wells that are being used in the run, click on Sample Type, and scroll to Unknown. 10. Change the dye layer to VIC, click on Sample Type, and scroll to Sample Type Setup. Click the Add button and scroll down to bottom row where New Control in the name column is indicated. Change the acronym to Unknown, change the name to Unknown, pick a color, change the reporter to VIC, and click OK when nished. 11. Click on Sample Type and scroll to Unknown. 12. If using either the 559/560 or 1088/1095-primer/probe combination for NAT1 genotype determination, repeat steps 10 and 11 using TET as the dye layer and reporter. 13. Click on Thermocycler conditions and in stage 3 of the program, change 95 C to 92 C for NAT1 genotype assays, change 60 C to 62 C for NAT2 genotype assays, and click OK.
The NAT1 PCR reactions begin with two initial hold steps (50 C for 2 min, followed by 95 C for 10 min) and 40 cycles of a two-step PCR (92 C for 15 sec, 60 C for 1 min). The NAT2 PCR amplication reactions begin with two initial hold steps (50 C for 2 min, followed by 95 C for 10 min) and 40 cycles of a two-step PCR (95 C for 15 sec, 62 C for 1 min).

14. Click on File, assign a name, and click Save. 15. Click the Show Analysis button and then the Run button.
The run will be completed in 2 hr. The uorescence intensity of each sample is measured at each temperature change to monitor amplication of the NAT1 or NAT2 gene. The nucleotide present at each SNP is determined by the uorescence ratio of the two SNP-specic uorogenic probes. The uorescence signal increases when the probe with the exact sequence match binds to the single-stranded template DNA and is digested by the 5 -3 exonuclease activity of AmpliTaq-Gold DNA polymerase (Applied Biosystems). Digestion of the probe releases the uorescent reporter dye (FAM, VIC, or TET) from the quencher dye (TAMRA).

Analyze completed run 16. Once the run is complete, click on Analysis and scroll to Analyze. Click OK.
This screen informs whether or not the samples were amplied by polymerase chain reaction (PCR). Amplication is evident by an increase in the relative number ( RN).

17. Click on File and scroll to Save. Close this run by clicking in the upper left-hand corner. 18. Click on File and scroll to New Plate. Change the plate type to Allelic Discrimination and click OK.
TaqMan-PCR Genotyping of NAT1 and NAT2

19. Highlight the wells that were used in the run. Click on Sample Type and scroll to Unknown.

4.15.6
Supplement 22 Current Protocols in Toxicology

Figure 4.15.1 SNP data from the 7700 Sequence Detector. The top graph illustrates SNP uorescence data for individual samples in the 96-well plate. The red circle cluster at upper left shows samples that are 1 or homozygous for one nucleotide (usually the more frequent one). The green circle cluster in the center shows samples that are 1/2 or heterozygous for the SNP. The blue circle cluster at lower right shows samples that are 2 or homozygous for the other nucleotide (usually the less frequent one). These values are imported into the bottom spreadsheet as described in the Basic Protocol. Thus, as shown in the database, the sample in well A-1 is in the green center cluster and is heterozygous for the two nucleotides; the sample in well A-2 is in the red upper left cluster and is homozygous for the common nucleotide; and the sample in well A-3 is in the blue cluster at lower right and is homozygous for the less common nucleotide. All cells in the 12th column (data illustrated as black squares) were negative controls (no DNA template). This black and white facsimile of the gure is intended only as a placeholder; for full-color version of gure go to http://www.currentprotocols.com/colorgures.

20. Click on the Show Analysis button. 21. Click on Post-PCR Read and let the instrument proceed until it is done. 22. Click on Analysis and scroll to Analyze. 23. Click on Analysis and scroll to Allelic Discrimination. Click on Normalize button and scroll to Dye Components. 24. Click on the magnifying glass and then click on the graph to bring the graph to scale.
There should be at least three identiable groups as illustrated in Figure 4.15.1.

25. Click on the lasso and circle the grouping in the upper left. Change the call to allele 1. Circle the grouping of Xs that are in the bottom right and change the call to allele 2. Circle the grouping of Xs that is in the middle and change the call to allele 1/2 . 26. Print the graph and the corresponding 96-well tray that contains the results. Click on File and scroll to Save As. Give the post-PCR read a name and click Save.
The results can be exported or copied to Excel or other programs for further analysis. The process of preparing the samples, preparing the 7700 Sequence Detector, and analyzing the run must be repeated for each SNP in human NAT1 and NAT2.
Techniques for Assessment of Chemical Transformation

4.15.7
Current Protocols in Toxicology Supplement 22

COMMENTARY Background Information

NAT1 and NAT2 encode N-acetyltransferase isozymes that catalyze the acetylation of many aromatic and hydrazine drugs as well as many aromatic and heterocyclic amine carcinogens present in the environment, industry, and the diet (Hein et al., 2000a; Butcher et al., 2002). Both NAT1 and NAT2 possess intronless, protein-coding, 870-bp exons. A related pseudogene, NATP, also has been identied (Vatsis et al., 1995). These NAT loci are separated by <500 kb in the orientation NAT1:NATP:NAT2 in the 8p22 region of chromosome eight (Upton et al., 2001). NAT1 and NAT2 share 87% nucleotide identity in the coding region, translating to only 55 amino acid differences (Vatsis et al., 1995; Hein et al., 2000a). NAT1 4 is dened as the reference allele and over 25 variant alleles have been identied in human populations (Table 4.15.1). The NAT1 alleles have genetic polymorphisms in both the coding and untranslated regions. The NAT1 10 allele, a common allele associated with rapid acetylator phenotype in some but not all studies (Hein et al., 2000a; Upton et al., 2001), possesses SNPs in the 3 -untranslated region. Many published genotype methods are designed only to distinguish a small subset of SNPs and do not distinguish NAT1 10 from NAT1 14A and other NAT1 alleles. Since NAT1 10 is often associated with environmental disease, it is particularly important that the NAT1 10 allele frequency be determined accurately. Several NAT1 alleles (NAT1 14, 15, 17, 19, and 22) are associated with reduced NAT1 catalytic activity and slow acetylator phenotype (Butcher et al., 1998; Hughes et al., 1998; Lin et al., 1998; Fretland et al., 2001b). Frequencies for some of the NAT1 and NAT2 alleles are rare reecting nucleotide diversity rather than genetic polymorphism (dened as frequency of at least 1%). NAT2 4 is dened as the reference allele and >30 NAT2 allelic variants have been identied in human populations (Table 4.15.2). The NAT2 variant alleles possess one or a combination of nucleotide substitutions in the NAT2 coding region. Seven of these: G191 A (R64 Q), C282 T (silent), T341 C (I114 T), C481 T (silent), G590 A (R197 Q), A803 G (K268 R), and G857 A (G286 E) have frequencies in human populations exceeding 1% depending upon the ethnic group (Hein et al., 2000a; Butcher et al., 2002). For example, the G191 A substitution common to the NAT2 14 gene cluster is relatively frequent in African populations (Delomenie et al., 1996; Loktionov et al., 2002), but it is virtually absent in Caucasian populations. NAT2 alleles possessing the C190 T (R64W), G191 A (R64Q), T341 C (I114T), G364 A (D122N), A411 T (L137F), A434 C (E145P), G590 A (R197Q), and/or G857 A (G286E) missense SNPs are associated with slow acetylator phenotypes (Leff et al., 1999; Fretland et al., 2001a; Hein, 2002; Svensson and Hein, in press). The NAT2 slow acetylator phenotypes are most likely not homogenous, but rather reect varying levels of slow acetylator phenotype due to different mechanisms among the various SNPs (Leff et al., 1999). The original NAT1 and NAT2 genotyping methods were designed to detect the most frequent SNPs (usually three or four) by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. These assays had the potential for genotype misclassications (Hein et al., 2000a; Deitz et al., 2004). Several improved PCR-RFLP genotyping assays for NAT1 and NAT2 polymorphism assay have been developed subsequently (Doll et al., 1995; Delomenie et al., 1996; Deitz et al., 1997; Lo-Guidice et al., 2000; Cascorbi et al., 2001; Vaziri et al., 2001; Loktionov et al., 2002). Although these assays are still frequently utilized for NAT1 and NAT2 genotyping, they are very labor intensive and incomplete digestion of the PCR products can lead to misinterpretation of the results (Cascorbi and Roots, 1999). Major advantages of the methods described here are that they do not require post-PCR processing (such as enzyme digestion) or the use of radioactivity. Since the methods amplify relatively small segments of NAT1 or NAT2, they are effective for human DNA samples derived from buccal cells or parafn-embedded tissues. In addition to other real-time PCRbased assays, alternative methods for determining NAT1 and NAT2 nucleotide polymorphisms include automated DNA sequencing, allele-specic oligonucleotide and enzymelinked immunosorbent assay (ELISA)-based oligonucleotide ligation assays (reviewed in Hein et al., in press).

Critical Parameters
Since NAT1, NAT2, and NATP are highly homologous, PCR primers must be specic for NAT1 or NAT2. Since most NAT1 and NAT2 alleles have multiple nucleotide

TaqMan-PCR Genotyping of NAT1 and NAT2

4.15.8
Supplement 22 Current Protocols in Toxicology

substitutions, it is important to phase their location on one or the other of the homologous chromosomes in the diploid genome (Cascorbi and Roots, 1999). Genotype misclassication also occurs when variant NAT alleles sharing a common nucleotide polymorphism are not distinguished. This should be avoided or minimized since it causes substantial bias in epidemiology investigations, particularly if geneenvironmental interactions are considered. All assays should be set up in a place that is clean and free of any plasmid or genomic DNA contamination. This method is very sensitive and will amplify even the smallest quantities of template DNA, so care must be taken to prevent contamination of foreign DNA. The 96-well plate can be cut to size if <96 samples are run in a single run. As soon as the DNA samples have been added to the appropriate wells, the optical caps should be placed on the optical tubes. When placing the optical caps on the optical tubes, make sure not to damage the optical caps in any way or it will affect the result of that sample. No-DNAtemplate amplication controls should be carried out to demonstrate no contamination of foreign DNA. Other controls that should be included are positive controls for each SNP. It is important that the DNA concentration be in the range of 5 to 25 ng/assay, however, much less DNA can be used for plasmid controls. The nucleotide substitutions at 559/560 and 1088/1095 are distinguished using a threeprobe system that has both nucleotide polymorphisms in the same probe. This is necessary because the 559/560 and the 1088/1095 substitutions are too close together to use the conventional two-probe TaqMan assays. The 559/560 and 1088/1095 polymorphisms are not analyzed the way the other polymorphisms are analyzed. To determine whether and how many polymorphisms exist in a sample, look at the increase in uorescence of the three probes. If only the FAM signal increases, then the sample is homozygous for 559C/560G or 1088T/1095C depending on which primer/probe set is used. If only the VIC signal increases, then the sample is homozygous for 559C/560A or 1088A/1095A depending on the primer/probe set used. If only the TET signal increases, then the sample is homozygous for 559T/560G or 1088T/1095A depending on which primer/probe set was used. Assignment of haplotype or allele requires phasing the SNPs. This may be ambiguous due to the overlapping nature of the nucleotide sub-

stitutions that occur among the allelic variants. In these instances, the entire NAT1 or NAT2 sequence should be amplied by PCR as previously described (Doll et al., 1995; Deitz et al., 1997). The PCR product is then cloned directly into pCR-TOPO (Invitrogen) following manufacturers instructions. This vector takes advantage of the 3 -A overhang added there by the Taq DNA polymerase during PCR. It also takes advantage of the topoisomerase gene that is covalently bound to the 3 -T overhang of the pCR-TOPO vector. This topoisomerase enzyme facilitates the direct ligation of the PCR product into the vector. Colonies possessing a single NAT1 or NAT2 allele can be used as a template for the genotyping assay or sequencing. A method to unambiguously assign the NAT2 genotype using allele-specic PCR combined with RFLP has been described (Delmonie et al., 1996).

Anticipated Results
Figure 4.15.1 illustrates the data generated by the 7700 Sequence Detector and its transfer to a database. The frequency of NAT1 and NAT2 SNPs, alleles, genotypes, and phenotypes differs markedly between ethnic groups (e.g., Delomenie et al., 1996; Upton et al., 2001; Doll and Hein, 2002; Loktionov et al., 2002).

Time Considerations
It is most efcient to test all samples for one SNP before proceeding on to the next SNP. However, when processing small numbers of samples, different SNPs can be done in a single run. It requires 2.5 hr for experienced users to assess one SNP in 96 samples. Preparation for each run can be accomplished in 15 min with use of master mixes and multi-channel pipets. Setting up the sequence detector takes 5 min. Once set up, each run takes 2 hr of instrument time. Following completion of the run, data transfer to the database can be accomplished in 10 min. For large experiments, economy of time and expense is achieved with instruments that can incorporate 384-well plates.

Literature Cited
Butcher, N.J., Boukouvala, S., Sim, E., and Minchin, R.F. 2002. Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J. 2:30-42. Butcher, N.J., Ilett, K.F., and Minchin, R.F. 1998. Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by C190T and G560A mutations. Pharmacogenetics 8:67-72.

Techniques for Assessment of Chemical Transformation

4.15.9
Current Protocols in Toxicology Supplement 22

Cascorbi, I. and Roots, I. 1999. Pitfalls in Nacetyltransferase 2 genotyping. Pharmacogenetics 9:123-127. Cascorbi, I., Roots, I., and Brockmoller, J. 2001. Association of NAT1 and NAT2 polymorphisms to urinary bladder cancer: Signicantly reduced risk in subjects with NAT1 10. Cancer Res. 61:5051-5056. Deitz, A.C., Doll, M.A., and Hein, D.W. 1997. A restriction fragment length polymorphism assay that differentiates human N-acetyltransferase-1 (NAT1) alleles. Anal. Biochem. 253:219-24. Deitz, A.C., Rothman, N., Rebbeck, T.R., Hayes, R.B., Chow, W-H., Zheng, W., Hein, D.W., and Garcia-Closes, M., 2004. Impact of misclassication in genotype-exposure interaction studies. Example of N-acetyltransferase 2 (NAT2), smoking, and bladder cancer. Cancer Epidem. Biomark. Prev. 13:1543-1546. Delomenie, C., Sica, L., Grant, D.M., Krishnamoorthy, R., and Dupret, J.M. 1996. Genotyping of the polymorphic N-acetyltransferase (NAT2 ) gene locus in two native African populations. Pharmacogenetics 6:177-185. Doll, M.A., Fretland, A.J., Deitz, A.C., and Hein, D.W. 1995. Determination of human NAT2 acetylator genotype by restriction fragmentlength polymorphism and allele-specic amplication. Anal Biochem. 231:413-420. Doll, M.A. and Hein, D.W. 2001. Comprehensive human NAT2 genotype method using single nucleotide polymorphism-specic polymerase chain reaction primers and uorogenic probes. Anal. Biochem. 288:106-108. Doll, M.A. and Hein, D.W. 2002. Rapid genotype method to distinguish frequent and/or functional polymorphisms in human N-acetyltransferase-1 (NAT1). Anal. Biochem. 301:328-332. Fretland, A.J., Leff, M.A., Doll, M.A., and Hein, D.W. 2001a. Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenetics 11:207-215. Fretland, A.J., Doll, M.A., Leff, M.A., and Hein, D.W. 2001b. Functional characterization of nucleotide polymorphisms in the coding region of human N-acetyltransferase 1. Pharmacogenetics 11:511-520. Hein, D.W. 2002. Molecular genetics and function of NAT1 and NAT2: Role in aromatic amine metabolism and carcinogenesis. Mutat. Res. 506-507:65-77. Hein, D.W., Doll, M.A., Fretland, A.J., Leff, M.A., Webb, S.J., Xiao, G.H., Devanaboyina, U.-S., Nangju, N.A., and Feng, Y. 2000a. Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidem. Biomarker Prev. 9:29-42. Hein, D.W., Doll, M.A., and Nerland, D.E., In press. Methods for N-acetyltransferase (NAT1 and NAT2) genotype determination. In Principles of Clinical Pharmacogenomics and Introduction to Pharmaco Proteomics (S.H.Y. Wong, M.W., Linder, and R. Valdes Jr., eds.), AACC Press, Washington, D.C.

Hein, D.W., Grant, D.M., and Sim, E. 2000b. Update on consensus arylamine N-acetyltransferase gene nomenclature. Pharmacogenetics 10:291292. Hughes, N.C., Janezic, S.A., McQueen, K.L., Jewett, M.A.S., Castranio, T., Bell, D.A., and Grant, D.M. 1998. Identication and characterization of variant alleles of human acetyltransferase NAT1 with defective function using paminosalicylate as an in-vivo and in-vitro probe. Pharmacogenetics 8:55-66. Leff, M.A., Fretland, A.J., Doll, M.A., and Hein, D.W. 1999. Novel human N-acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype. J. Biol. Chem. 274:34519-34522. Lin, H.J., Probst-Hensch, N.M., Hughes, N.C., Sakamoto, G.T., Louie, A.D., Kau, I.H., Lin, B.K., Lee, D.B., Lin, J., Frankl, H.D., Lee, E.R., Hardy, S., Grant, D.M., and Haile, R.W. 1998. Variants of N-acetyltransferase NAT1 and a casecontrol study of colorectal adenomas. Pharmacogenetics 8:269-281. Lo-Guidice, J.-M., Allorge, D., Chevalier, D., Debuysere, H., Faxio, F., Latte, J.-J., and Broly, F. 2000. Molecular analysis of the Nacetyltransferase 1 gene (NAT1 ) using polymerase chain reaction-restriction fragmentsingle strand conformation polymorphism assay. Pharmacogenetics 10:293-300. Loktionov, A., Moore, W., Spencer, S.P., Vorster, H., Nell, T., ONeill, I.K., Bingham, S.A., and Cummings, J.H. 2002. Differences in Nacetylation genotypes between Caucasians and Black South Africans: Implications for cancer prevention. Cancer Detection Prev. 26:15-22. Svensson, C.K., and Hein, D.W., In press. Phenotypic and genotypic characterization of Nacetylation. In Drug Metabolism and Transport: Molecular Methods and Mechanisms (L.H. Lash, ed.) The Humana Press, Totowa, N.J. Upton, U., Johnson, N., Sandy, J., and Sim, E. 2001. Arylamine N-acetyltransferases- of mice, men and microorganisms. Trends Pharmacol. Sci. 22:140-146. Vatsis, K.P., Weber, W.W., Bell, D.A., Dupret, J.M., Price-Evans, D.A., Grant, D.M., Hein, D.W., Lin, H.J., Meyer, U.A., Relling, M.V., Sim, E., Suzuki, T., and Yamazoe, Y. 1995. Nomenclature for N-acetyltransferases. Pharmacogenetics 5:1-17. Vaziri, S.A.J., Hughes, N.C., Sampson, H., Darlington, G., Jewett, M.A.S., and Grant, D.M. 2001. Variation in enzymes of arylamine procarcinogen biotransformation among bladder cancer patients and control subjects. Pharmacogenetics 11:7-20.

Key References
Butcher et al., 2002. See above. Review of the role of NAT1 and NAT2 polymorphisms on drug response. Doll and Hein, 2001. See above. Original report of NAT2 genotype method described in this unit.

TaqMan-PCR Genotyping of NAT1 and NAT2

4.15.10
Supplement 22 Current Protocols in Toxicology

Doll and Hein, 2002. See above. Original report of NAT2 genotype method described in this unit. Hein et al., 2000a. See above. Review of the role of NAT1 and NAT2 polymorphisms on cancer risk. Hein et al., In press. See above. Review of different NAT1 and NAT2 genotyping methods. McQueen, C.A. 2001. Measuring the activity of arylamine N-acetyltransferase (NAT). In Current Protocols in Toxicology., 4.6.1-4.6.13. John Wiley & Sons, New York. Describes common protocols for measuring Nacetyltransferase catalytic activity.

Internet Resources
http://www.louisville.edu/medschool/ pharmacology/NAT.html Website for NAT1 and NAT2 alleles.

Contributed by David W. Hein and Mark A. Doll University of Louisville School of Medicine Louisville, Kentucky

Techniques for Assessment of Chemical Transformation

4.15.11
Current Protocols in Toxicology Supplement 22