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Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles

Leukemia volume 18, pages 255–266 (2004)Cite this article

Abstract

Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor–recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor–recipient pairs. Samples from nine of the donor–recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

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Acknowledgements

We thank Raul Figueroa and David Fange for their excellent technical assistance with array printing and Excel macros, respectively. Lovisa Lovmar and Tomas Axelsson provided valuable comments on the manuscript.

This work was supported by grants from the Vårdal Foundation, the Swedish Research Council (VR) and the Knut and Alice Wallenberg Foundation (WCN).

Author information

Author notes

  1. M Fredriksson, G Barbany, U Liljedahl and A-C Syvänen: Contributed to the appendix section.

Authors and Affiliations

  1. Department of Medical Sciences, Uppsala University, Uppsala, Sweden

    M Fredriksson, U Liljedahl & A-C Syvänen

  2. Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden

    G Barbany & M Hermanson

  3. National Public Health Institute, Helsinki, Finland

    M Kataja

Authors
  1. M Fredriksson
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  2. G Barbany
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Corresponding author

Correspondence to A-C Syvänen.

Appendix: Method in Focus

Appendix: Method in Focus

Assay characteristics

In the primer extension reaction (minisequencing), a DNA polymerase is used to extend a detection primer, which anneals immediately adjacent to a single-nucleotide polymorphism (SNP), with labeled nucleotide analogues.22,43 For solid-phase minisequencing in the microtiter plate format, a DNA fragment containing the SNP is amplified using one biotinylated and one nonbiotinylated PCR primer. The biotinylated PCR products are captured on streptavidin-coated microtiter plate wells and denatured. The nucleotides at the SNP site are identified in the immobilized DNA by primer extension reactions, in which a DNA polymerase incorporates a [3H]dNTP. The results of the assay are numeric counts per minute (c.p.m.) values expressing the amount of [3H]dNTP incorporated in the minisequencing reactions. For minisequencing in the tag-microarray format, the SNPs are amplified in multiplex PCR reactions and the PCR products from one individual are combined and subjected to multiplex minisequencing reactions with four fluorescently labeled ddNTPs. Minisequencing detection primers with 5′ tag sequences are used, and the products of the cyclic minisequencing reactions performed in solution are captured on microarrays carrying oligonucleotides complementary to the tag sequences. The incorporated ddNTPs are detected by scanning the microarray slide at four different wavelengths.30 In both assay formats, the ratio (R) between the radioactive or fluorescent signals corresponding to the two SNP alleles are used for assigning the genotypes of the SNPs and for quantitative determination of the SNP allele fractions in mixed samples.

Here, we present a two-phased minisequencing strategy for monitoring chimerism after stem cell transplantation (SCT). First, informative SNPs with alleles differing between donor and recipient are identified using the multiplex microarray-based minisequencing system. This format allows the analysis of 12 samples and two controls for up to 60 SNPs in duplicate in both polarities of the DNA. Secondly, the development of chimerism is followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by the minisequencing method in the microtiter plate format. This format allows duplicate analysis of two SNPs in 12 samples or three SNPs in eight samples per 96-well microtiter plate.

Protocol

DNA preparation

DNA is extracted from the samples according to a standard procedure. The DNA concentration is measured spectrophotometrically at 260 nm and the isolated DNA is stored at −20°C.

SNPs

The SNPs to be genotyped should be selected from different chromosomal locations to ensure that they are inherited independent of each other. To be of maximum informative content they should have allele frequencies near 0.5 (range 0.3–0.7) in the population of interest. The SNPs can be validated by analyzing pooled DNA samples using quantitative minisequencing in a microtiter plate format as described below for chimerism analysis.

PCR amplification

For the microarray-based genotyping, 6 ng of genomic DNA is amplified in multiplex PCR reactions with up to 10 primer pairs in each reaction or in single PCRs according to a standard PCR protocol, for example, with 0.03 U/μl of AmpliTaq Gold DNA polymerase, 3 mM MgCl2, 200 μ M dNTPs and primer concentrations ranging from 0.2 to 0.3 μ M in 50 μl of buffer supplied with the enzyme.

For genotyping in microtiter plates, the SNP markers are amplified in individual reactions using 10 ng genomic DNA, 0.2 μ M of the 5′ biotinylated PCR primer, 1.0 μ M of the nonbiotinylated primer according to a standard PCR protocol, for example, with 1.0 U AmpliTaq Gold DNA polymerase, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 200 μ M dNTPs, in a final volume of 100 μl.

Preparation of microarrays

The capturing oligonucleotides, complementary to the tag sequence of the minisequencing primers, are covalently attached via their 3′-NH2 groups to CodeLink™ activated slides according to the manufacturer's instructions. This chemistry has been shown to be optimal for our method.44 The oligonucleotides are immobilized in duplicate spots of 150 μm in diameter to form 14 subarrays per slide in an ‘array of arrays’ conformation32 using a contact printing robot such as a ProSys 5510A instrument with Stealth Micro Spotting Pins (SMP3B). A Cy3-labeled oligonucleotide is included as a spotting control and one capture oligonucleotide is included in the subarray to be used to capture a TAMRA-labeled hybridization control. The remaining amino-reactive groups on the slides are blocked with ethanolamine according to the manufacturer's instructions.30

Genotyping on microarrays

The PCR products from each individual are combined and concentrated to about 30 μl using spin dialysis or by another suitable method. An aliquot of 14 μl of the concentrated PCR product is treated with 0.5 U/μl Exonuclease I and 0.1 U/μl shrimp alkaline phosphatase in 5.6 mM MgCl2 and 50 mM Tris-HCl, pH 9.5, in a total volume of 21 μl at 37°C for 60 min, followed by inactivation of the enzymes at 95°C for 15 min in a thermocycler. Each minisequencing reaction mixture contains 21 μl of enzyme-treated PCR product, the tagged minisequencing primers at 5.0 nM each, 0.18 μ M of fluorescently labeled TexasRed-ddATP, TAMRA-ddCTP, R110-ddGTP, and 0.27 μ M Cy5-ddUTP, 0.017% Triton X-100, 17 mM Tris-HCl, pH 9.5 and 0.07 U/μl of Thermo Sequenase™ DNA Polymerase. The total minisequencing reaction volume is 30 μl and the cyclic reactions are performed in a thermocycler for 99 cycles of 95°C and 55°C for 20 s each.

The arrayed slides are preheated to 42°C in a reaction rack with a silicon rubber grid forming 14 separate reaction chambers on each slide.31,32 The hybridization mixtures, containing 30 μl of minisequencing reaction product and 0.4 nM TAMRA-labeled hybridization control oligonucleotide in 44 μl of 900 mM NaCl, 90 mM sodium citrate, pH 7.0, are added to each reaction chamber on the slide. The hybridization time is 2.0–2.5 h at 42°C, after which the slides are rinsed briefly with 600 mM NaCl and 60 mM sodium citrate, pH 7.0, at room temperature. The slides are then washed twice for 5 min with 300 mM NaCl and 30 mM sodium citrate, pH 7.0 and 0.1% SDS preheated to 42°C and twice for 1 min with 30 mM NaCl and 3 mM sodium citrate, pH 7.0, at room temperature. Finally, the slides are dried by centrifugation for 5 min at 500 r.p.m.

Signal detection and genotype assignment

Fluorescence signals are measured with an array scanner equipped with four lasers. We use a ScanArray® 5000 instrument with the excitation lasers Blue Argon 488 nm, Green HeNe 543.8 nm, Yellow HeNe 594 nm and Red HeNe 632.8 nm. The signal intensities are measured with the QuantArray® analysis 3.1 software. Background correction is carried out by measuring fluorescence intensities from 10 spots below the subarray and by subtracting the average background from every spot in the same subarray. The genotypes are determined by calculating the ratio (R) between the signal intensities of the two alleles for each SNP, for example, by using a Microsoft Excel™ macro.

Genotyping using solid-phase minisequencing

Four 10 μl aliquots of the individual PCR product and 40 μl of phosphate-buffered saline with 0.1% Tween 20 are added to streptavidin-coated microtiter plate wells and incubated for 1.5 h at 37°C with shaking. The plates are washed six times with 40 mM Tris-HCl, pH 8.8, 1 mM EDTA, 50 mM NaCl and 0.1% Tween 20 in a plate washer, after which the nonbiotinylated strand of the PCR product is removed by denaturation with 60 μl 50 mM NaOH for 3 min. The microtiter plates are then washed as above before adding 50 μl of minisequencing reaction mix containing DNA polymerase buffer, 0.05 U AmpliTaq DNA polymerase, 0.05 μCi of the appropriate [3H]dNTP ([3H]dATP TRK 633, 69 Ci/mmol; [3H]dCTP TRK 625, 57 Ci/mmol; [3H]dGTP TRK 627, 33 Ci/mmol; [3H]dTTP TRK 576, 111 Ci/mmol) and 10 pmol of the appropriate detection primer to each well. The plates are incubated for 10 min at 50°C. After washing as above, the detection primers are released with 100 μl 50 mM NaOH and the incorporated [3H]dNTP is measured in a liquid scintillation counter.

Determination of SNP allele fractions and proportion of donor cells

The samples are analyzed in duplicate and the average signals are used. The signal ratio (R) between the two possible [3H]dNTPs to be incorporated at each SNP site is used. For each SNP, the signal ratios in the donor and patient samples prior to SCT and the follow-up samples after SCT are compared to the corresponding signal ratios in heterozygous samples (Rhet) where the two SNP alleles are present in equal amounts. Using Rhet as reference value for the quantitative determination, the SNP allele fractions are calculated according to the formula:

where R=c.p.m.Allele1/c.p.m.Allele2

The determination of the proportion of donor cells in the follow-up samples is based on the allele fraction of the unique SNP that distinguishes between the donor and the recipient in each pair. The signal from the unique SNP allele represents all the contribution from the differing individual in the case of homozygosity, but only half in the case of heterozygosity. In the latter case, the proportion of donor cells is determined by multiplying the fraction of the unique SNP allele by two.

Time requirement

The total time required for the PCR amplification is about 3 h, which includes a hands-on time of approximately 30 min, provided that DNA has been extracted and that premade master mixes are used. The minisequencing procedure for one microarray slide with up to 14 samples requires a total time of 7 h including a hands-on time of approximately 3 h. Scaling up to process three microarray slides in parallel does not increase the required time, except for the processing of data, which will requires approximately 1 h per microarray slide. For minisequencing in a microtiter plate format, the total time for processing one microtiter plate with 12 samples is 4.5 h including a hands-on time of 1 h.

Sensitivity

The sensitivity for detecting an SNP allele present as a minority in a mixed DNA sample ranges from 0.5 to 2.5% depending on the SNP marker (Figure 3 in the Main Article). The detection limit has been determined in experiments using mixtures of DNA with known genotypes and with a total amount of 10 ng of genomic DNA in each reaction. The sensitivity of detection of the minority allele for an SNP marker is influenced by the nucleotide sequence flanking the SNP, which affects the specificity of nucleotide incorporation by the DNA polymerase. The sensitivity is also influenced by the specific activity and number of [3H]dNTPs incorporated in the reaction. The sensitivity of detecting chimerism can thus be improved by selecting favorable SNPs for the analysis.

Reproducibility and accuracy

The mean values for the coefficients of variance for the points in the standard curves (Figure 3 in the Main Article) are 8%, and vary between 6 and 10% between the three SNPs. The method is highly accurate with a linear relationship between the logarithm of the minsequencing ratios and the logarithm of ratios between the two alleles subjected to the analysis, with regression coefficients (R2) higher than 0.99 for the standard curves.

Cost of the assay

The overall cost per analysis depends on the total number of samples analyzed in parallel. The approximate costs given here are the costs for one sample processed in parallel with 12 samples. The reagent cost for a single PCR is 0.4 Euro. For the minisequencing on microarrays, the reagent costs per sample are 4.4 Euro (51 SNP genotypes in duplicate in both DNA polarities). For the minisequencing in the microtiter plate format, the cost per sample is 1 Euro (one SNP genotype in duplicate). Laboratory equipment and staff are not included in the calculation.

Troubleshooting

For minisequencing on microarrays

False-positive signals

  • Hairpin loop secondary structures involving the 3′-end of a primer can give rise to template-independent primer extension. To avoid this problem, evaluate the primer sequences with a primer design program that predicts secondary structures.

Low signals

  • Cy5-ddUTP has lower incorporation efficiency than the other ddNTPs, which can give rise to lower signals. Higher concentrations of Cy5-ddUTP can be used to compensate for this phenomenon.

High background

  • Background problems can arise if the hybridization chamber is not kept humid and the sample dries out on the slide.

Minisequencing in a microtiter plate

False-positive signals

  • The presence of other dNTPs than the intended [3H]dNTP during the minisequencing reaction will cause unspecific extension of the detection step primer. It is important to remove completely all dNTPs from the PCR by washing the microtiter plate wells thoroughly after the capturing step.

Low signals

  • The [3H]dNTPs used as detectable groups in the solid-phase minisequencing method have low specific activities, therefore it is important that the PCR amplification has been efficient. Make sure that 1/10 of the PCR product is visible after agarose-gel electrophoresis and staining with ethidium bromide.

  • The biotin binding capacity of the microtiter plate wells has been exceeded. The binding capacity of the streptavidin-coated microtiter well is 2–5 pmol of biotinylated oligonucleotide. Therefore, the amount of biotinylated primer used for PCR should be adjusted not to exceed this capacity.

Materials

Hardware

  1. 1

    Microcentrifuge.

  2. 2

    Spectrophotometer.

  3. 3

    Access to contamination-free PCR facilities.

  4. 4

    Multichannel pipet or microtiter plate washer.

  5. 5

    Thermocycler.

  6. 6

    Access to oligonucleotide synthesis.

  7. 7

    Access to microarray manufacturing.

  8. 8

    A reaction rack for microarry slides, for example one with a silicon rubber grid prepared as described in Pastinen et al32 and Lindroos et al31.

  9. 9

    Thermoblock or water bath with a shaker.

  10. 10

    Liquid scintillation counter (1414, Wallac, Turku, Finland).

  11. 11

    Array scanner with software for signal analysis (we use a ScanArray® 5000 instrument with QuantArray® analysis 3.1 software, Perkin-Elmer, Life Science, Boston, MA, USA).

Consumables

  1. 1

    Microcon YM-30 device (eg Millipore Corporation, Bedford, MA, USA).

  2. 2

    Microscope glass slides (CodeLink™ Activated Slides, Amersham Biosciences, Uppsala, Sweden).

  3. 3

    Microtiter plates with streptavidin-coated wells (eg Combiplate 8, Labsystems, Helsinki, Finland).

Reagents and solutions

For minisequencing on microarrays
  1. 1

    Oligonucleotides: 3′ amino-modified oligonucleotide primers with a spacer sequence of 15T residues in their 3′-end. Fluorescently labeled hybridization control oligonucleotide, complementary to one of the spotted tag oligonucleotides on the microarray.

  2. 2

    Exonuclease I 10 U/μl (USB Corporation, Cleveland, OH, USA).

  3. 3

    Shrimp alkaline phosphatase 1 U/μl (USB Corporation, Cleveland, OH, USA).

  4. 4

    Thermo Sequenase™ DNA Polymerase (Amersham Biosciences, Uppsala, Sweden).

  5. 5

    Fluorescently-labeled TexasRed-ddATP, TAMRA-ddCTP, R110-ddGTP and Cy5-ddUTP (Perkin-Elmer, Life Sciences, Boston, MA, USA).

For minisequencing in a microtiter plate format
  1. 1

    Oligonucleotides: one PCR primer of each pair is biotinylated at its 5′-end. Minisequencing detection primer.

  2. 2

    AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA).

  3. 3

    [3H]-labeled dNTPs: [3H]dATP, TRK 633; [3H]dCTP, TRK 625; [3H]dGTP, TRK 627; [3H]dTTP, TRK 576 (Amersham Biosciences, Uppsala, Sweden).

  4. 4

    Scintillation reagent (for example, Hi-Safe II, Wallac, Turku, Finland).

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Fredriksson, M., Barbany, G., Liljedahl, U. et al. Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles. Leukemia 18, 255–266 (2004). https://doi.org/10.1038/sj.leu.2403213

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  • DOI: https://doi.org/10.1038/sj.leu.2403213

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