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Characterization of (CA)n Microsatellite Repeats from Large‐Insert Clones
Publication Name:
Current Protocols in Human Genetics
Unit Number:
UNIT 2.4
DOI:
10.1002/0471142905.hg0204s00
Online Posting Date:
May, 2001 Abstract
The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit determination of sequences flanking the microsatellites. When cosmids or large-insert phage clones are used as primary sources of (CA)n repeat markers, they have traditionally been subcloned into plasmid vectors such as pUC18 or M13 mp18/19 cloning vectors to obtain fragments of suitable size for DNA sequencing. This unit presents an alternative approach whereby a set of degenerate sequencing primers that anneal directly to (CA)n microsatellites can be used to determine sequences that are inaccessible with vector-derived primers. Because the primers anneal to the repeat and not to the vector, they can be used with subclones containing inserts of several kilobases and should, in theory, always give sequence in the regions directly flanking the repeat. Degeneracy at the 3 end of each of these primers prevents elongation of primers that have annealed out-of-register.The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit.
Materials
Basic Protocol: Sequencing of (CA)n Repeats Using Degenerate Primers
Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
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Cosmid or phage clone containing insert DNA with (CA)
n repeats - Restriction endonucleases with 4- and 6-base recognition sites and appropriate buffers
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Hybridization buffer, prewarmed to 65°C (see recipe)
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100 pmol/µl (CA)
15 oligonucleotide probe (see recipe) -
10 mCi/ml [-
32 P]ATP (3000 mCi/mmol) -
Low-stringency wash solution: 6× SSC/0.1% (w/v) SDS, prewarmed to 65°C
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High-stringency wash solution: 0.2× SSC/0.1% (w/v) SDS, prewarmed to 65°C
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100 pmol/µl (CA)
11 (A/G/T) and (GT)11 (A/C/T) degenerate sequencing primers (see recipe) -
AmpliTaq Cycle sequencing system (Perkin-Elmer Cetus) or equivalent reagents (cpmb unit 7.4A)
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Thermal cycler (Perkin-Elmer Cetus GeneAmp PCR System 9600 or equivalent)
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Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), Southern blotting (unit 2.7), 5¢-end labeling of oligonucleotides (appendix 3E), measuring radioactivity by TCA precipitation (appendix 3E), subcloning (cpmb unit 3.16), preparation of phage DNA or plasmid minipreps for dideoxy sequencing (cpmb unit 7.3), and colony hybridization (unit 2.3)CAUTION: [-
32 P]ATP is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
Looking for materials? Suppliers Guide
Figures
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Figure 2.4.1Template and three degenerate primers used in the sequencing reaction. The template has a (CA)n repeat bordered by a G immediately 5¢ to the repeat. In a mixture of three primers, all containing (GT)11 but differing in their 3¢ terminal base, only the primer that has a C at its 3¢ end (and is therefore perfectly matched) will be efficiently extended by DNA polymerase and give rise to a sequence ladder. -
Figure 2.4.2(A) Sequence ladder produced by thermal cycle sequencing of cosmid cCI11-475 with a (CA)11 (A/G/T) degenerate primer mix. The dotted region marked A (5¢-CCACTCCTCAGAAGATC-3¢) was used to design a unique complementary sequencing primer (primer A). (B) Sequence ladder produced by using this primer to sequence back across the (CA)n repeat. (C) PCR amplification of 12 unrelated individuals using primer A and a second unique primer designed from the results shown in part B (primer B). The four left-most lanes show an M13 sequence ladder used as a molecular weight marker. The right-most lane shows the PCR product from cosmid cCI11-475.
Literature Cited
| Literature Cited | |
| Browne, D.L. and Litt, M. 1992. Characterization of (CA) | |
| Craxton, M. 1991. Linear amplification sequencing, a powerful method for sequencing DNA. Methods 3:1. | |
| Church, G.M. and Kieffer-Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185-188. | |
| Key Reference | |
| Browne, D.L. and Litt, M. 1992. See above | |
| Original description of this methodology. | |
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