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Method and Technology

A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice

Sung-Ryul Kim1, Jungil Yang2, Gynheung An2, Kshirod K. Jena1,*
Plant Breeding and Biotechnology 2016;4(1):99-106.
Published online: February 28, 2016

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*Corresponding author: Kshirod K. Jena, k.jena@irri.org, Tel: +63-2-580-5600, Fax: +63-2-891-1236
• Received: January 4, 2016   • Revised: January 31, 2016   • Accepted: February 4, 2016

Copyright © 2016 The Korean Society of Breeding Science

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A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice
Plant Breed. Biotech.. 2016;4(1):99-106.   Published online February 28, 2016
DOI: https://doi.org/10.9787/PBB.2016.4.1.99
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A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice
Plant Breed. Biotech.. 2016;4(1):99-106.   Published online February 28, 2016
DOI: https://doi.org/10.9787/PBB.2016.4.1.99
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A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice
Image Image Image Image Image
Fig. 1 Agarose gel image of leaf extracts and screening of the optimal dilution degree of the crude leaf extracts for polymerase chain reaction (PCR). (A) Gel image of leaf extracts. Leaf tissue (4 cm long) was collected from 15 to 80 days old plants of varieties NSIC Rc222 (lane 1), PSB Rc82 (lane 2), Nipponbare (lane 3), and Osmancik-97 (lane 4), respectively. Following extraction, the supernatants (15 μl) were electrophoresed in 1% agarose gel. (B) PCR amplification efficiency according to dilution degree of leaf extracts. Leaf tissue (2 cm long) was prepared from 50 days-old plant of NSIC Rc222 (lane 1) and Taichung 65 (lane 2) as described. PCRs were performed with four primer sets. Product sizes and GC contents are shown next to the gel images. CTAB-extracted DNA was used as a control. M: DNA size marker, CTAB: cetyltrimethylammonium bromide; 500 ng of genomic DNA (gDNA) extracted by CTAB method, T: 100 mM tris-HCl pH 9.5, P: 1 M KCl, E: 10 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0.
Fig. 2 The influences of sample quantity and plant developmental stages on polymerase chain reaction (PCR) of tris-phosphate (TPE)-extracted DNA. Leaf tissue (0.5, 1, 2, 4, and 8 cm long) was collected from the seedling stage (SD, 20 days after germination) of IR24 and NSIC Rc222 and from the grain filling stage (GF, 10 days after pollination) of Taichung 65 and IR64, respectively. DNAs were prepared by TPE buffer with six times dilution. PCR efficiency was tested using Chr06A (A), Chr02A (B), Chr07A (C), and Chr03B (D) primers. Cetyltrimethylammonium bromide (CTAB)-extracted DNA was used as control. M: DNA size marker.
Fig. 3 DNA quality test by polymerase chain reaction (PCR) amplifications of regions of various GC content (34% to 67%) and product sizes (580 to 1,987 bp). The leaf extracts were prepared based on the tris-phosphate protocol from 11 rice accessions. PCRs were performed with five primer sets. M: DNA size marker.
Fig. 4 DNA stability test. Leaf extracts were prepared by the tris-phosphate protocol from 12 BC2F2 plants of breeding line YPF14-853, and then they were stored at 4°C for 8 months. Genomic DNA integrity was tested by gel electrophoresis of 15 μl of the stored extracts (A) and by polymerase chain reaction amplification of the stored extracts with Chr09A primer set (B). M: DNA size marker.
Fig. 5 Various polymerase chain reaction (PCR) applications using genomic DNA extracted by the TPE method. (A) Genotyping of 12 T2 plants of the rice T-DNA insertional mutant line, PFG_3A-52268. (B) F1 hybridity test using rice microsatellite (RM) marker, RM 11 from 10 F1 plants. (C) Discrimination of SNP type DNA polymorphism for marker-assisted breeding. The A/G SNP located in the promoter region of Gn1a gene which regulates grain number per panicle (Ashikari et al. 2005) was determined from 12 BC1F2 plants of the intermediate breeding line YPF14-582 using the tetra-primer PCR method (Ye et al. 2001). M: DNA size marker, p1 and p2: parents of F1.

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A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice

List of polymerase chain reaction primers used in this study.

PrimersZ) Forward primer (5′→3′) Reverse primer (5′→3′)
Chr02A TTGACAACCACTCCTGTCCT CCCTTCAACATGGTTGAGGT
Chr02B TGGGCATGTTTCTGGAGACA CTCGAATGGCTTCCAATGAC
Chr03A GCTCCAAGAACTAATACAAGC CTAGCTTGAGGGTTCCTGCA
Chr03B TTGGCTTGATTTCCTGTGCTA GCTTTCTGCTCCTGCTGTAA
Chr03C TGCACCATAACTACACTTGCT CTTGAATGCTTGCTGGTCGA
Chr06A TTCCCATCTGCACTACCATAATCC GAGCAGAGATGTGCTTTGCTACC
Chr06B GAGATCAGTACTTGTACTAGC TCAACTTACTCCCTCAGTCT
Chr07A GACCTCACCTGCTATAGCTA GCTCGATCGAGCCGATCAT
Chr08A GGTTGTTCAGTGGCAATGTC GCAAAAGTGCAGCTAACCAC
Chr09A GCAAGTGCTCACCCAAGTG AGCAACCACTGAGACAGCAT

Z)The number in the primer name represents rice chromosome number.

Table 1 List of polymerase chain reaction primers used in this study.

The number in the primer name represents rice chromosome number.

Table 1

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