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Automated Fluorescent Genotyping

Jeff Hall¹, Elizabeth Nanthakumar¹

¹Sequana Therapeutics, Inc., La Jolla, California

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 2.8

DOI:

10.1002/0471142905.hg0208s14

Online Posting Date:

May, 2001

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Abstract

With the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand to undertake large-scale genotyping projects. This unit describes the basic methodology, optimization of markers, and allele calling.With the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand.

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Unit Introduction
Basic Protocol: PCR Amplification of SSLPs for Automated Fluorescent Genotyping
Support Protocol 1: Pooling Fluorescently Labeled PCR Products for Genotype Analysis
Support Protocol 2: Gel Electrophoresis of Pooled PCR Products
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: PCR Amplification of SSLPs for Automated Fluorescent Genotyping

Materials

Patient DNA samples at a concentration of 4 ng/µl in TE buffer
10× PCR buffer (Perkin-Elmer)
10 mM 4dNTP mix (appendix 2D or Perkin-Elmer)
25 mM MgCl₂
20 µM fluorochrome-labeled forward primer for each microsatellite marker
20 µM reverse primer for each microsatellite marker
5 U/µl AmpliTaq Gold DNA polymerase (Perkin-Elmer)

96-well plates (Robbins)
Drying oven
Multichannel pipettor
96-well strip caps (Robbins) or 96-well plastic plate-sealing film (Costar)
5-ml and 50-ml tubes
Reagent trays, sterile
Thermal cycler(s) equipped to handle 96-well plates

Support Protocol 1: Pooling Fluorescently Labeled PCR Products for Genotype Analysis

Materials

Fluorochrome-labeled PCR products (see Basic Protocol)
4× BB loading dye: 0.167% (w/v) bromphenol blue/30% (v/v) glycerol
50 µg/ml DNA size standard (e.g., DNA Mass Ladder, Life Technologies)
Genescan 500 Tamra size standard (Perkin-Elmer)
Beckman CS-6 centrifuge and Beckman TS-2000 rotor (or equivalent) with 96-well plate adaptors
96-well plates
Multichannel pipettor
96-well plastic plate-sealing film (Costar)
90°C oven
Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol 2: Gel Electrophoresis of Pooled PCR Products

Materials

Gel-loading plate (see Support Protocol 1)
6% (w/v), 24-cm well-to-read acrylamide gel (see recipe)
1× TBE electrophoresis buffer (appendix 2D)
Automated fluorescent sequencer (e.g., ABI 373 or 377 for four-color detection, Perkin-Elmer; or Pharmacia, LICOR for single-color detection) with computer and mouse
Genescan 672 Collection, Analysis, and Genotyper software (Perkin-Elmer)
60-cc syringe with needles
Lane-indicator strip, plastic (Perkin-Elmer; optional)
Micropipettor
Gel-loading micropipettor tips

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Figures

Figure 2.8.1

Multiplex of thirteen microsatellite markers from chromosomes 15 and 16 in a single gel lane. (A) Genescan Analysis electropherogram view of a single lane. (B) Genotyper view of automated allele assignments for three markers, labeled with different dyes, in an overlapping size range. The bottom traces show the internal size standard.

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Figure 2.8.2

Scans showing typical allele patterns for dinucleotide (A) and tetranucleotide (B) microsatellite repeats.

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Figure 2.8.3

Scans showing effects of stutter on dinucleotide repeat allele patterns. (A) Heterozygote with alleles separated by 10 bp. (B) Heterozygote with alleles separated by 4 bp. (C) Heterozygote with alleles separated by 2 bp. (D) Homozygote.

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Figure 2.8.4

Scan showing decreased amplification of the larger (higher-molecular-weight) allele.

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Figure 2.8.5

Scan showing allele dropout of higher- or lower-molecular-weight alleles. (A) Original Genotyper allele assignment with the larger (higher-molecular-weight) dropout allele uncalled; the edited Genotyper allele assignment is below. (B) Original Genotyper allele assignment of an unusual peak pattern with the smaller (lower-molecular-weight) dropout allele uncalled; the edited Genotyper allele assignment is below.

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Figure 2.8.6

Scans showing sizing error caused by missing 50-bp size standard. (A) Genotyper file with the marker and size standard aligned by size. The missized allele is not detectable in this view. (B) Genotyper file with the marker and size standard aligned by scan number. It is now easily observed that the allele size is incorrect because this peak migrated faster than the 75-bp standard. (C) Expanded view showing the beginning of the analysis file. The lack of 50-bp size standard in the analysis file which caused the sizing error is now apparent.

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Figure 2.8.7

Scans showing effect of underpooling sample. (A) Adequately pooled sample. (B) Underpooled sample showing decreased signal-to-noise ratio.

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Figure 2.8.8

Scans showing effect of overpooling sample. (A) Overpooled Tet marker exhibiting signal bounce-back at the 169-bp allele and spectral bleedthrough in the other channels. (B) Overpooled Fam marker with bleedthrough interfering with the 100-bp size standard.

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Figure 2.8.9

Scans showing examples of good (A) and poor (B) peak quality.

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Figure 2.8.10

Scans showing effect of primer tailing on peak quality and allele binning. (A) Original allele peak pattern exhibiting plus A (notice the humps on the right side of each allele peak) and the corresponding allelogram for this marker across five 96-well plates. (B) Allele peak pattern after tailing the 5¢ end of the unlabeled primer with the sequence 5¢-GTTTCTT-3¢ and the corresponding allelogram (notice the easily identified distinct allele bins).

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Literature Cited

Literature Cited
	Beckmann, J.S., Tomfohrde, J., Barnes, R.I., Williams, M., Broux, O., Richard, I., Weissenbach, J., and Bowcock, A.M. 1993. A linkage map of human chromosome 15 with an average resolution of 2 cM and containing 55 polymorphic microsatellites. Hum. Mol. Genet. 2:2019-2030.
	Brownstein, M.J., Carpten, J.D., and Smith, J.R. 1996. Modulation of non-templated nucleotide addition by Taq polymerase: Primer modifications that facilitate genotyping. BioTechniques 20:1004-1010
	Diehl, S.R., Ziegle, J., Buck, G.A., Reynolds, T.R., and Weber, J.L. 1990. Automated genotyping of human DNA polymorphisms. Am. J. Hum. Genet. 47:A177.
	Litt, M. and Luty, J.A. 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44:397-401.
	Smeets, H.J.M., Brunner, H.G., Ropers, H.H., and Wieringa, B. 1989. Use of variable simple sequence motifs as genetic markers: Application to study of myotonic dystrophy. Hum. Genet. 83:245-251.
	Weber, J.L. and May, P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Gen. 44:338-396.