Optimization of Annealing Temperature, Concentration of Primer, and Genome DNA for Amplification of Disease Resistance Gene Markers on Tiger Shrimp (Penaeus monodon)

  Optimization of Annealing Temperature, Concentration of Primer, and Genome DNA for Amplification of Disease Resistance Gene Markers on Tiger Shrimp (Penaeus monodon) _{1 2 2} Andi Tenriulo , Ahyar Ahmad , Abd. Rauf Patong ₁ Email: anditenriulo@yahoo.com Chemistry Graduate, Science Faculty, Hasanuddin University, Makassar, South Sulawesi, ₂ 90245 Lecturer of Chemistry Department, Science Faculty, Hasanuddin University, Makassar, South Sulawesi, 90245

  Abstrak. Identifikasi udang tahan penyakit secara molekuler dengan teknik PCR merupakan salah satu langkah awal dalam upaya perakitan strain udang windu Penaeus monodon tahan penyakit. Penelitian ini bertujuan untuk mengoptimalisasi proses PCR agar memperoleh gen target yang sesuai dan meningkatkan produk amplifikasi. Isolasi genom DNA dilakukan dengan metode fenol-kloroform. Amplifikasi fragmen mikrosatelit menggunakan kit RTG-PCR beads dengan primer spesifik MsatPm-F dan MsatPm-R. Optimalisasi suhu annealing diatur pada 50,

52, 54, dan 56 ºC. Optimalisasi konsentrasi primer dilakukan dengan konsentrasi 0,5 µM;

1,0 µM; 1,5 µ M; dan 2,0 µ M primer forward dan reverse, dan DNA genom yang digunakan sebagai cetakan masing-masing sebanyak 0,5; 1,0; 1;5; dan 2,0 µg/µL. Hasil penelitian menunjukkan bahwa kondisi reaksi PCR sangat menentukan proses amplifikasi untuk identifikasi udang windu tahan penyakit, dimana suhu annealing 54 ºC, konsentrasi primer MsatPm-F dan MsatPm-R 1 µM DNA genom sebanyak 1 µg/µ L memberikan amplifikasi terbaik karena menghasilkan target yang sesuai, pita yang tebal, dan tidak terjadi mispriming. kata kunci: Annealing, DNA genom udang windu, konsentrasi primer, optimalisasi, PCR.

  Abstract. Identification of the molecular basis of disease-resistant shrimp with PCR technique is one of the initial steps in Assembly of tiger shrimp Penaeus monodon strains resistant to the disease.This study aims to optimize the PCR process in order to obtain the corresponding target genes and increases the amplification products. Isolation of genomic DNA was conducted by using phenol-chloroform method. Amplification of microsatellite fragments using RTG-PCR beads kit with specific primers MsatPm-F and MsatPm-R. Optimization of annealing temperature is set at 50, 52, 54, and 56 ºC. Optimization of primer concentration is done with a concentration of 0,5 µ M; 1,0 µM; 1,5 µM; dan 2,0 µM forward and reverse primer, and genomic DNA were used as templates, respectively 0.5; 1.0; 1,5; and 2.0 µ g/µ L. The results showed that the conditions of the PCR reaction largely determine the amplification process for the identification disease resistance gene marker of tiger shrimp, where annealing temperature 54 ºC, the concentration 1 µM of Primer MsatPm-F and MsatPm-R, and genomic DNA as much as 1 µg/µL provide best amplification because it produces the corresponding target, thick ribbon, and not occur mispriming. ₁ Keywords : annealing, genomic DNA tiger shrimp, optimization, PCR, primer concentration.

  Corresponding author to E-mail: andi.tenriulo@yahoo.co.id

  INTRODUCTION

  Development in Bioinformatics and the technique of polymerase chain reaction (PCR) can be used as an approach to develop a specific marker. PCR method is used to amplify specific regions of the DNA chain. Short chain oligonucleotide primer designed to hybridizy each end of the DNA chain at the target and then extend it on the opposite side of the DNA. To design a required primary data gene sequences that encode proteins similar to the gene to be amplified by PCR.

  One important factor that affects the quality of PCR-based molecular detection is the selection of the appropriate primer (Yowono, 2005). PCR primers are oligonucleotides that act as initiation of DNA molecule amplification. The existence of the PCR primers, the target gene will be amplified during the PCR reaction. PCR amplification requires specific primers (sequences specific oligonukelotida), usually consists of 10-20 nucleotides and is designed based on the conserved region of the genome. The longer the primer, the more specific the region amplified. Designing a good primer should consider some specific rules, which shows the amount of amplicon, primer length, melting point or Tm, and do not form secondary structures such as dimers, cross-dimer, or hairpin (Dale and Schantz, 2002). The content of the oligonucleotide primer will determine the annealing temperature of the PCR reaction.

  Mukherjee and Mandal (2009) have used DNA microsatellite markers to identify disease resistance of tiger shrimp with observed two populations tiger shrimp that are resistant to disease and can not stand white spot disease virus (WSSV, White Spot Syndrome Virus). The results indicate a difference in microsatellite DNA fingerprinting is very significant. In disease-resistant population, found only one microsatellite DNA band 317 bp, whereas the population is not resistant disease, there is an additional bands at 71 bp. Band at 71 bp was found in populations only disease resistance. The discovery of DNA markers are resistant black tiger shrimp and WSSV disease resistance to disease, serve as the basis of DNA markers to determine disease resistance in tiger shrimp.

  The concentration of DNA, primer length, base composition of primer, the concentration of Mg ions, and primer hybridization temperature should be carefully controlled in order to obtain DNA bands were intact and good. Optimization of the PCR process is necessary to produce the desired character. So far the optimization of PCR conditions for detection of DNA markers of disease resistance in black tiger shrimp has not been reported. Because it is necessary to develop methods, especially on the optimization of PCR conditions and optimization of the concentration of genomic DNA and primers to obtain disease resistance marker method on the corresponding tiger shrimp.

MATERIALS AND METHODS

  Isolation of Genomic DNA

  Extraction of genomic DNA of tiger shrimp is done using phenol-chloroform method that has been developed in grouper (Parenrengi et al., 2010). Muscle/meat from the pleopod 25-50 mg taken individually and aseptically, then preserved with 250 µL TNES using urea buffer in 1.5 µL eppendorf tubes (Asahida et al., 1996).

  To determine the success of the extraction method used, a mixture of 3 µL genomic DNA and 1.0 µ L of loading dye electrophoresed in a 0.8% agarose gel in 1 x TBE (Tris-Boric acid-EDTA) at a voltage of 50 volt. Gel stained by adding 1.0 uL red gel solution (1 µL/30 mL agarose), and as the standard marker used DNA marker λ/Hind III.

  Once the shrimp genomic DNA was isolated, the next process is the detection of DNA target. Detection sequences of DNA target performed by amplification of microsatellite DNA fragments using PCR machine GenAmp 7200 (Applied Biosystems). Specific primers designed based on information from the Gene Bank accession number AF077565. Amplification was performed using PCR Kit PureTaq Ready To Go (RTG) beads. This kit contains 2.5 units of Taq polymerase, 10 mM Tris-HCl pH 9, 50 mM KCl, 1.5 mM MgCl

  2

  , and 200 µM dNTP- mix.

  Optimization Of Annealing Temperature

  PCR conditions optimized annealing temperature at 50, 52, 54, and 56 ºC. PCR is conditioned by: Pre-denaturation 94 °C for 3 minutes, followed by 35 cycles of (denaturation 94 °C 30 second , annealing 50, 52, 54, and 56 ºC, extension 72 ºC for 1 minute), and final extension 72 ºC for 3 minutes.

  Microsatellite analysis to be used refers to the method that has been developed by Mukherjee and Mandal (2009). Analysis were performed with indicators of the presence of fragments at position 317 bp and 71 bp. Data presented by descriptive.

RESULTS AND DISCUSSION

  50 ρmol/µL) and DNA genome as a template, respectively 0.5 µg; 1.0 µg; 1.5 µ g; and 2.0 µg (stock concentration 50 µg/µL). The concentration of primer and genomic DNA fragments that show good will be used for subsequent amplification process.

  Analysis of Microsatellite DNA Markers of Disease Resistant

  Optimization of primer concentrations and genomic DNA performed to obtain the desired target gene. To determine the concentration of primer and the appropriate genomic DNA used various concentrations of primer concentration of 0,5 µM; 1,0 µM; 1,5 µM; dan 2,0 µM forward and reverse (stock concentration of primer is

  Optimizing Concentrations of Primer and Genomic DNA

  Genomic DNA Extraction

  Genomic DNA was isolated from black tiger shrimp using phenol-chloroform method. Visualization of genomic DNA extraction results shown in Figure 1 .

  Figure 1. Profiles of genomic DNA

  isolated from black tiger shrimp (1-10),

  DNA Marker λ/Hind III (M); arrows indicate the position of genomic DNA fragments.

  Results documentation on agarose gel showed a single fragment of DNA at position 23 130 bp, marked with band genomic DNA clean without background. This indicates that the DNA is not degraded and contaminated, and indicating that preservation solution TNES-urea and phenol-chloroform method is a method that can be used for genomic DNA isolation tiger shrimp. The results of measurements of the concentration and purity of tiger shrimp genomic DNA shown in Table 1.

  Amplification fragment of microsatellite sequences performed using PCR with specific primers MsatPm-F and MsatPm-R are designed based on information from the Gene Bank accession number AF077565.1. PCR reactions using RTG-PCR kit pureTaq beads. PCR results are then separated using electrophoresis techniques . To know that the amplification of the target DNA fragments were successful, 1 µL PCR product electrophoresed on 2% agarose gel at a voltage of 50 volts for 1 hour and documented with a Gel Documentation System . The molecular weight of the DNA fragments were determined by using Low Range DNA Marker.

  Table 1. The concentration and purity of

  ratio is less than 1.8 then there is contamination of the protein, whereas the phenol contamination and other organic materials are characterized by high values ratio of OD

  The results showed that optimization of annealing temperature at a temperature of 54 ºC, primers can amplify the target DNA with the thickness of the band is not too different. At a temperature of 50 ºC and 52 ºC also occurred amplification yet seen the band a little bit thin on the lower targets, while at a temperature of 56 ºC did not appear the band at position 300 bp due to the higher temperatures the bond between the primer and the DNA template is unstable so there is no amplification ( Figure 2 ).

  microsatellite DNA markers disease resistance of tiger shrimp in optimization of annealing temperature 50 ºC (1-5), 52 ºC (6-10), 54 ºC (11-14), and 56 ºC (16-20). The arrows indicate the position of the target DNA fragment. M: Low Range DNA Marker

  Figure 2. The results of the analysis of

  Annealing is one of the PCR process, where the primer will recognize and attach to the DNA target sequence. Annealing process on the DNA thread that has been open requires optimum temperature. Annealing temperatures used are generally 5 ºC lower than the melting temperature (Tm) (Dale and Schantz, 2002). Tm values can be calculated by multiplying the 2 ºC for each nucleotide adenine and thymine and 4 ºC for each nucleotide guanine and cytosine. The results show the optimization on the temperature 54 °C resulted in DNA bands clearer with a specific size other than the annealing temperature. Temperatures that are too high can lead to amplification does not occur. Instead the temperature is too low causing the primer attached to the side of the DNA that is not the homologs, the consequences can be amplified many areas are not specific in its genome.

  Optimization of Annealing Temperature

  more higher than 2.0. The quality and quantity of DNA is very influential on the success of the PCR process. Low purity would inhibiting primer composition of bases in the DNA chain. Small size can reduce DNA primer template. Similarly, the amount of DNA that is too high will affect the results of amplification (Surandar and Imron, 2010).

  280

  /OD

  260

  280

  genomic DNA of tiger shrimp

  /OD

  260

  ratio ranged from 1.8 to 2.0. If the DNA molecule with OD

  280

  /OD

  260

  116,02 ± 27,4 Genomic DNA isolated from black tiger shrimp showed good purity as suggested by Linacero et al., (1998) which states that the good purity of the genomic DNA marked with OD

  1,91 ±0,04

  genomic DNA

  Sample Purity Concentratio n (µg/mL)

  In the DNA molecule, hydrogen bonds influence to form the helical structure between the paired strands. Pairing between nucleotides adenine (A) and Thymine (T) is determined by the presence of two hydrogen bonds, while the guanine (G) and cytokine (C) is determined by three hydrogen bonds. Because it is necessary to separate the G-C bond needed higher temperatures. The percentage content of G and C determine the Tm (melting temperature) of a primer, which is the temperature at which the majority of double-stranded DNA separates into single strands. Tm values will determine primer annealing temperature needed to attach to the template DNA (Yowono, 2009).

  According to Dale and Schantz (2002), the annealing process on the DNA thread is already open requires optimum temperature, because the temperature is too high can lead to amplification does not occur or the temperature is too low otherwise the primer cause attached to the other side of the genome that are not homologs, consequently can not be amplified many specific areas in the genome. Annealing temperature is determined based on the primers used are influenced by the length and composition of the primer .

  Sambrook et al. (1998) states that the temperature settings greatly affect the process of amplification in a PCR reaction, because it will result in increase exponentially specific fragments of DNA targets. Products produced in the first cycle will act as a template in the next cycle. Tm values that are too low can cause the primer does not work at high temperatures, whereas the Tm values that are too high can cause the primer is not in the target area (mispriming) so that the products are not specific .

  Optimizing of Primer Concentration

  Use primers with a concentration of 1 µM amplification results were better than the other concentrations. The use of higher concentrations of the primer causes fragments of the target DNA at position 300 bp becomes thinner even at concentrations 3 µM and 4 µM looks very thin.

  Similar results were reported by Abidin (2005), and Surandar and Imron (2010) that the use of high concentrations of the Primer causes not amplified fragment the target at high molecular weight. Muladno (2002) stated that the Primer concentration in the reaction is too high can cause primer attached to the 3 end is to complement.

  Figure 3. The results of the analysis of

  microsatellite DNA markers in disease resistance of tiger shrimp optimization of primer concentration. Note: 1 (1 µg/µL), 2 (2 µ g/µ L), 3 (3 µ g/µ L), 4 (4 µ g/µ L), M (geneRuler Low Range DNA markers). The arrows indicate the position of the target DNA fragment.

  Optimization of Genomic DNA Concentration

  The concentration of genomic DNA allow the probability of amplification process. Primer will recognize appropriate target DNA that occur subsequent elongation, but the occurrence of rapid cycle DNA strands will make illegible or not recognizable perfectly, resulting in little fragments at a certain size and can also lead to misprime. The results of genomic concentration optimization show differences concentrations showed no difference in the pattern of fragments striking , especially in the higher molecular weight. However, at lower concentrations more clearly visible bands on the low bp (Figure 4). This suggests that the use 1 µg/µL of the genome DNA is considered the best for amplified the desease resistent gene marker.

  Abidin (2005) reported that the lower DNA concentration showed no obvious banding in the position of higher molecular weight, in contrast to the higher concentration of DNA bands were not clear on the position of the lower molecular weight. In Yowono (2005), the recommended concentration of genomic DNA present in the reaction mixture with primer concentrations between 0.1-0.5 µM is 0.01 µg/µL.

  Figure 4. The results of the microsatellite

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  REFERENCES

  This research was funded by the state budget of DIPA Research Institute for Coastal Aquaculture (RICA) Maros , Fiscal Year 2012 . Thanks go to the entire staff of researchers and technicians Biotechnology Laboratory RICA Maros, which has supported the implementation of this study.

  ACKNOWLEDGEMENTS

  PCR reaction conditions determine the amplification process for the identification of disease-resistant tiger shrimp, where the optimal annealing temperature at 54 ºC, 1 µM primers MsatPm-F and MsatPm-R primer concentration and genomic DNA as much as 1 µg/µL gave the best amplification for generating the appropriate target, thick band, and does not happen mispriming.

  CONCLUSION

  1 (1 µg/mL), 2 (2 µg/mL), 3 (3 µg/mL), 4 (4 µ g/mL), and M (geneRuler Low Range DNA markers ). The arrows indicate the position of the target DNA fragment.

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