PCR Procedure for the Isolation of Trinucleotide Repeats

Abstract

Microsatellites, also referred to as short tandem repeats (STR) or simple sequence repeats (SSR), are highly polymorphic and abundant sequences dispersed throughout most eukaryotic nuclear genomes (1-3). In recent years, microsatellites have been used for linkage map construction, population genetics, molecular evolution studies, forensic sciences, and as parentage testing markers (4,5).

1 Introduction

Microsatellites, also referred to as short tandem repeats (STR) or simple sequence repeats (SSR), are highly polymorphic and abundant sequences dispersed throughout most eukaryotic nuclear genomes (1-3). In recent years, microsatellites have been used for linkage map construction, population genetics, molecular evolution studies, forensic sciences, and as parentage testing markers (4,5).

Trinucleotide repeats, such as (CAG)_n and (CGG)_n repeats, have drawn increasing attention in various aspects of both human gene mapping and clinical genetics. Genes with trinucleotide repeats, such as transcription-regulatory proteins and homeobox genes, are frequently found in mammalian genomes (6). Some of these genes are involved in neuropsychiatric disorders in human, such as Huntington’s disease (7) and spinocerebellar ataxia type 1 (8). At present, the function of the trinucleotide repeats is not clear.

Polymorphism of trinucleotide repeats can be resolved more easily in alleles differing by one repeat than those of dinucleotide repeats. Thus, tri- and tetranucleotide repeats are useful for human forensic sciences. The abundance and polymorphic information of trinucleotide repeats in the human genome have been characterized by Gastier et al. (9). In the human genome, (CAG)_n repeats occur, on average, once every 4400 kb, making them less abundant than dinucleotide repeats. Thus, it is considered that the construction of an enrichment library is useful for the isolation of trinucleotide repeats.

A novel, rapid, and convenient cloning method by the construction of an enrichment library has been developed for the isolation of trinucleotide repeats in human genome, and applied for the isolation of tri- and dinucleotide repeats in equine genome (10-14). The method includes the following procedures: adapter PCR of genomic DNA, enrichment procedure, adapter PCR for large preparation, followed by cloning. The enrichment procedure includes the following steps: capturing some other element from genomic DNA by hybridization to biotin-labeled probes in solution, a nucleotide substrate-biased polymerase reaction, a subsequent extraction with magnetic beads coated with streptavidin. In particular, the nucleotide substrate-biased polymerase reaction is useful for the isolation of microsatellites containing long repeating units. The method is designed to select only the longer repeats. The advantage of the method is the efficient isolation of long repeat-containing highly polymorphic microsatellites, because microsatellites greater than 12-repeats in length are more likely to be polymorphic (15).

In addition, this method offers the major advantage that it can be repeated any number of times for enrichment, indicating that the method is useful for the cloning of less frequent microsatellites, such as trinucleotide repeats.

This chapter describes the construction of an enrichment library for the microsatellite-isolation. The method described here is a detailed version of a method that have been reported previously (10-14). The method is outlined in Fig. 1 .

Fig. 1.

A schematic diagram of the construction of a library enriched for microsatellites. The first line represents the Sau3AI-digested genomic DNA and the adapter sequences. The striped boxes are micro satellite target sequences, SA denotes the streptavidin-coated magnetic particles and B indicates biotin molecules.

2 Materials

• 1. Autoclaved sterile 1.5-mL tube.

• 2. Autoclaved sterile 200 µL-PCR tube.

• 3. Purified genomic DNA.

• 4. Sau3AI (Takara Shuzo).

• 5. 10X buffer for Sau3AI: 500 mM Tris-HCl, pH 7.5, at 25°C, 100 mM MgCl₂, 10 mM dithiothreitol, 1000 mM NaCl.

• 6. Mung bean nuclease (Takara Shuzo).

• 7. 1X buffer for Mung Bean nuclease: 30 mM acetate, pH 5.0, at25°C, 100 mM NaCl, 1 mM zinc acetate, 5% glycerol.

• 8. Autoclaved, distilled, deionized water.

• 9. Oligonucleotide primers for adapter ligation. For first enrichment: Primer (1) 5′-GCACTCTCCAGCCTCTCAGTGCAG-3′ (100 µM and 25 µM). Anti primer (1) 5′-GATCCTGCACTG-3′ (100 µM). For second enrichment: Primer (2) 5′-AGCACTCTCCAGCCTCTCACCGAG-3′ (100 µM and 25 µM). Anti primer (2) 5′-GAT CCT CGG TGA-3′ (100 µM).

• 10. Biotin-labeled oligo (CAG)₈ (20 µM).

• 11. T4 DNA ligase (Takara Shuzo).

• 12. 10X ligase buffer: 660 mM Tris-HCl, pH 7.6, at 25°C, 66 mM MgCl₂, 100 mM dithiothreitol, 1 mM ATP.

• 13. rTaq polymerase (5 U/µL) (Takara Shuzo).

• 14. 10X rTaq buffer (MgCl₂ free): 100 mM Tris-HCl, pH 8.3, at 25°C, 500 mM KCl.

• 15. ExTaq polymerase (5 U/µL) (Takara Shuzo).

• 16. 10X ExTaq buffer (MgCl₂ plus) (Takara Shuzo).

• 17. LATaq polymerase (5 U/µL) (Takara Shuzo).

• 18. Perfect Match PCR enhancer (Stratagene, CA).

• 19. An enzyme mixture: 0.5 µL of rTaq polymerase, 0.2 µL of 10X diluted LATaq polymerase, and 2 µL of Perfect Match PCR enhancer (see Note 5 ).

• 20. dNTP (each 2.5 mM of dATP, dCTP, dGTP, and dTTP).

• 21. d3NTP (each 2.5 mM of dATP, dCTP, and dGTP).

• 22. 1X binding/washing buffer: 1 M NaCl, 10 mM Tris-HCl, pH 8.0, at 25°C, 1 mM EDTA.

• 23. 2X binding/washing buffer: 2 M NaCl, 10 mM Tris-HCl, pH 8.0, at 25°C, 1 mM EDTA.

• 24. Alkaline buffer: 1 mM NaOH, 10 mM Tris, 1 mM EDTA.

• 25. CotI DNA.

• 26. Streptavidin MagneSphereR Paramagnetic Particle (Promega, WI). Wash by adding and removing 0.5X SSC at room temperature twice, and resuspend in 100 µL of 1X binding/ washing buffer with CotI DNA (100 ng/µL).

• 27. Magna-Sep (a magnetic stand) (Gibco BRL, MD).

• 28. TE buffer: 10 mM Tris-HCl, pH 8.0, at 25°C, 1 mM ethylenediaminetetraacetic acid (EDTA).

• 29. Phenol equilibrated in TE buffer.

• 30. Chloroform:isoamyl alcohol: 24:1, v/v.

• 31. Glycogen (20 µg/µL) (Roche, Mannheim, Germany).

• 32. 3 M Sodium acetate.

• 33. 5 M Ammonium acetate.

• 34. 100%Ethanol.

• 35. 70%Ethanol.

• 36. G-50 Spin column (Roche, Mannheim, Germany).

• 37. T-vector (Promega, WI).

• 38. Competent Escherichia coli (E. coli) cells for electroporation.

3 Methods

3.1 Adapter PCR of Genomic DNA

The steps described in this method correspond to step I and step II of Fig. 1 .

• 1. Add in the following order: approx 20 µg of genomic DNA, 10 µL of 10X buffer for Sau3AI, 5 µL of Sau3AI, and up to 100 µL of sterile water. Incubate at 37°C overnight. Add 100 µL of sterile water.

• 2. Extract by adding 100 µL of phenol that has been preequilibrated with TE buffer. Vortex. Add 100 µL of chloroform: isoamyl alcohol. Vortex. Centrifuge in a microfuge at room temperature for 30 s to separate the phases. Remove the upper (aqueous) phase (approx 200 µL) and place in a fresh sterile 1.5-mL tube.

• 3. Repeat step 2 once.

• 4. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% ethanol. Incubate at -80°C for at least 10 min.

• 5. Recover the restricted genomic DNA by centrifugation in a microfuge (12 000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 400 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 µL of TE buffer (see Note 1 ).

• 6. In a sterile 1.5-mL tube, add in the following order: 500 ng of the restricted genomic DNA, each 2.5 µLof 100 µM primer (1) and antiprimer(1), 3 µL of 10X buffer for ligase, and up to 30 µL of sterile water.

• 7. Incubate at 53°C for 10 min in a heat block.

• 8. Incubate the tube with the heat block in refrigerator at 4°C for 60 min.

• 9. Add 1 µL of ligase, and incubate at 12°C overnight.

• 10. Add 470 µL of sterile water.

• 11. In a sterile 200-µL PCR tube, add in the following order: 10 µL of the ligation mixture, 5 µL of 20 µM primer (1), 10 µL of 10X buffer for ExTaq polymerase (MgCl₂ plus), 8 µL of each 2.5 mM dNTP, 0.5 µL of ExTaq polymerase, and 66.5 µL of sterile water.

• 12. Prepare four tubes for the sample.

• 13. Amplify by PCR using the following cycle profiles: initial denaturation at 72°C for 5 min; approx 20 cycles of 1 min each at 94°C and 72°C; and then 10 min at 72°C for final extension (see Note 2 ).

• 14. Extract by adding 200 µL of phenol that has been preequilibrated with TE buffer. Vortex it. Add 200 µL of chloroform:isoamyl alcohol. Vortex and centrifuge it in a microfuge at room temperature for 30 s to separate the phases. Remove the upper phase (approx 400 µL) and place in a fresh sterile 1.5-mL tube.

• 15. Repeat step 14 once.

• 16. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% ethanol. Incubate at -80°C for at least 10 min.

• 17. Recover the PCR products by centrifugation in a microfuge (12 000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 600 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min.

• 18. Resuspend the pellet in 50 µL of 1X mung bean nuclease buffer.

• 19. Add 1 µL of mung bean nuclease. Incubate at 37°C for 45 min. Add 150 µL of sterile water.

• 20. Extract by adding 100 µL of phenol that has been preequilibrated with TE buffer. Vortex. Add 100 µL of chloroform: isoamyl alcohol. Vortex and centrifuge it in a microfuge at room temperature for 30 s to separate the phases. Remove the upper phase (approx 200 µL) and place in a fresh sterile 1.5-mL tube.

• 21. Repeat step 20 once.

• 22. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% ethanol. Incubate at -80°C for at least 10 min.

• 23. Recover the PCR products by centrifugation in a microfuge (12 000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 600 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 µL of TE buffer.

• 24. Purify the PCR product by passing the remaining excess primers through a G-50 spin column by centrifugation (see Notes 1 and 3).

3.2 Enrichment Procedure

The steps described in this method correspond to step III, step IV, and step V of Fig. 1 .

• 1. In a sterile 200-µL PCR tube, add in the following order: 500 ng of the purified PCR products, 1 µL of 20 µM biotinylated oligo-(CAG)₈, 10 µL of 10X buffer for rTaq polymerase (MgCl₂ free), 10 µL of 15 mM MgCl₂, 8 µL of each 2.5 mM d3NTP, and up to 100 µL of sterile water. As negative controls, prepare two reaction mixtures without the purified PCR products or the biotinylated oligo-(CAG)₈, respectively.

• 2. Denature the sample at 94°C for 3 min to make target microsatellites accessible to the probe, and incubate it for each 5 min at 80°C and 75°C, respectively, to hybridize the biotinylated oligo-(CAG)₈ to the denatured PCR products.

• 3. Add 2.7 µL of enzyme mixture to the reaction solution kept at 75°C above (see Note 4 ).

• 4. Incubate it at 77°C for 10 min to carry out the nucleotide substrate-biased polymerase reaction.

• 5. Chill on ice, then move all the solution to a fresh 1.5-mL tube on ice which contains 200 µL of 2X binding/washing buffer to stop the polymerase reaction.

• 6. Add 100 µL of streptavidin-coated magnetic beads in the presence of 100 ng/µL of CotI DNA as the competitor.

• 7. Incubate the sample at 37°C for 30 min (see Note 5 ).

• 8. Capture the magnetic bead-complexes with a magnet stand, and then remove the supernatant containing excess unbound oligos and noncomplementary sequences from the tube.

• 9. Wash the sample by adding 500 µL of 1X binding/washing buffer at room temperature. Gently pipet the sample. Capture the magnetic bead-complexes with the magnet stand. Remove the supernatant.

• 10. Repeat step 9 twice.

• 11. Wash by adding 100 µL of 1X binding/washing buffer at 80°C. Gently pipet the sample. Capture the magnetic bead-complexes with the magnet stand. Remove the supernatant.

• 12. Repeat step 11 once.

• 13. Elute ssDNAs, which were containing repeat sequences, by adding and incubating 50 µL of alkaline buffer at 80°C from the biotinylated oligo-(CAG)₈. Gently pipet the sample. Capture the magnetic beads with the magnet stand. Recover the supernatant to a fresh 1.5-µLtube.

• 14. Repeat step 13 once.

• 15. Precipitate the eluted ssDNAs by adding 0.5 µL of glycogen (20 µg/µL), 40 µL of 5 M ammonium acetate, vortex briefly, and then 400 µL of cold 100% ethanol. Incubate at -80°C for at least 15 min.

• 16. Recover the ssDNAs by centrifugation in a microfuge (12 000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 600 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min. Resuspend the pellet in 20 µL of TE buffer.

3.3 Adapter PCR for Large Preparation of the Repeat Sequences

The step described in this method corresponds to step VI of Fig. 1 .

• 1. In a sterile 200-µL PCR tube, add in the following order: 2 µL of the resuspended ssDNAs, 2 µL of 20 µM primer (1), 5 µL of 10X buffer for ExTaq polymerase (MgCl₂ plus), 4 µL of each 2.5 mM dNTP, 0.25 µL of ExTaq polymerase, and 36.75 µL of sterile water. Also prepare reaction mixtures of the two resuspended products without the purified PCR products or the biotinylated oligo-(CAG)₈, respectively (see Note 6 ).

• 2. Amplify by PCR using the following cycle profiles: initial denaturation at 94°C for 2 min; 15 cycles of 1 min each at 94°C and 72°C; and then 10 min at 72°C for final extension.

• 3. As a preliminary experiment, confirm the number of cycles of the second PCR procedure for large preparation. In three fresh sterile 200-µL PCR tubes, add in the following order: 10 µL of the first PCR products of sample, the negative control without the purified PCR products, or the negative control without the biotinylated oligo-(CAG)₈ as templates, 4 µL of 20 µM primer (1), 10 µL of 10X buffer for ExTaq polymerase (MgCl₂ plus), 8 µL of each 2.5 mM dNTP, 0.5 µL of ExTaq polymerase, and 67.5 µL of sterile water.

• 4. Amplify the sample DNA by PCR using the following cycle profiles: initial denaturation at 94°C for 2 min; approx 13-18 cycles of 1 min each at 94°C and 72°C; and then 10 min at 72°C for final extension.

• 5. Take 5-10 µL aliquot of the PCR to visualize the products by agarose gel electrophoresis (1.5% agarose). Confirm the number of cycles of the second PCR that does not amplify PCR products of the two negative controls.

• 6. After the confirmation, amplify the first PCR products by PCR using the same condition and the confirmed cycle profiles.

• 7. Prepare and amplify four tubes of the PCR products with the condition of step 6.

• 8. Extract by adding 200 µL of phenol that has been preequilibrated with TE buffer. Vortex. Add 200 µL of chloroform:isoamyl alcohol. Vortex. Centrifuge in a microfuge at room temperature for 30 s to separate the phases. Remove the upper phase (approx 400 µL) and place in a fresh sterile 1.5-mL tube.

• 9. Repeat step 9 once.

• 10. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% ethanol. Incubate at -80°C for at least 10 min.

• 11. Recover the PCR products by centrifugation in a microfuge (12 000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 600 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 µL of TE buffer.

• 12. Purify the PCR product by passing the remaining PCR reaction through a G-50 spin column by centrifugation (see Note 1 ).

3.4 Second Cycle for Enrichment

The step described in this method corresponds to step I of Fig. 1 .

• 1. When the second enrichment procedure was desired, add in the following order: 5 µg of the first enriched PCR products, 10 µL of 10X buffer for Sau3AI, 5 µL of Sau3AI, and up to 100 µL of sterile water. Incubate at 37°C overnight (see Note 7 ). Add 100 µL of sterile water.

• 2. Extract by adding 100 µL of phenol that has been preequilibrated with TE buffer. Vortex the sample. Add 100 µL of chloroform:isoamyl alcohol. Vortex and centrifuge it in a microfuge at room temperature for 30 s to separate the phases. Remove the upper phase (approx 200 µL) and place in a fresh sterile 1.5-mL tube.

• 3. Repeat step 2 once.

• 4. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% ethanol. Incubate at -80°C for at least 10 min.

• 5. Recover the PCR products by centrifugation in a microfuge (12,000g) at 4°C for 20 min. Discard the supernatant and wash the pellet with 400 µL of cold 70% ethanol. Carefully discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 µL of TE buffer.

• 6. Purify the PCR product by passing the remaining PCR through a G-50 spin column by centrifugation.

• 7. In a sterile 1.5-mL tube, add in the following order: 250 ng of the purified PCR products, each 2.5 µL of 100 µM primer (2) and antiprimer (2), 3 µL of 10X buffer for ligase, and up to 30 µL of sterile water.

• 8. Incubate at 53°C for 10 min in the heat block.

• 9. Incubate the tube with the heat block at 4°C for 60 min.

• 10. Add 1 µL of ligase, and incubate at 12°C overnight.

• 11. Add 70 µL of sterile water.

• 12. Repeat steps 3.1.3-3.1.5, 3.2, and 3.3 for second cycle enrichment.

3.5 Construction of Enrichment Library

The PCR products enriched for (CAG)_n repeats should be constructed by direct cloning into a T-vector using T4 DNA ligase, taking advantage of the 3′-A overhangs often produced by Taq polymerase. These recombinants should be transformed into competent E. coli cells by electroporation (see Note 8 ).

4 Notes

• 1. Take an aliquot containing 500 ng of genomic DNA or the PCR products to check the enrichment rate of microsatellites by Southern blot analysis (see Note 8 ).

• 2. With the adapter PCR procedure, PCR conditions are optimized to generate a smear of the PCR products without specific bands. If some specific bands appear, the adapter sequences (primer 1 or primer 2) should change to new designed adapter sequences. The enrichment rate of microsatellites would be influenced by the generation of specific bands. By increasing amount of the ligation mixtures, the number of cycles for PCR could be reduced. It is possible that the reduced cycles of the PCR might get rid of the specific bands by the adapter primers.

• 3. Checkthe absence of excess primers by agarose gel electrophoresis. If the excess primers are visualized with the electrophoresis, the methods to purify them (see Subheading 3.1.14-24) should be repeated until the absence of the excess primers. The presence of the excess primers decreases the efficiency of the nucleotide substrate-biased polymerase reaction.

• 4. Prepare the fresh enzyme mixtures to prevent the inactivation of the enzymes.

• 5. Gently mix the reaction tubes, because the magnetic beads precipitate during the incubation.

• 6. The confirmation of the number of cycles of PCR for large preparation should be carried out for every lot of the experiments. The PCR with superfluous cycles might reduce the enrichment rate of microsatellites.

• 7. For this method to be repeated, the primer for the second enrichment must be changed from that for the first enrichment. The adapter change prevented amplification from the first primer. Amplification with alternative sets of primers would then produce different specific libraries of sequences complementary to a common target.

• 8. To analyze the enrichment rate, we performed Southern blot analysis of each product. The results reveal that the enrichment cloning method achieved 10²-fold enrichment by the first round procedure. Finally, 10²-³-fold enrichment was achieved for (CAG)_n repeat by the second round. A potential risk of the second round of PCR was considered, because a single DNA fragment was amplified to produce many copies. The results obtained in the previous study, however, indicated that only about 10% of the clones isolated were identical (11).