Application of PCR-RFLP techinique in genotyping for RS165599 polymorphism on COMT gene

To establish and complete a protocol for genotyping of single polymorphism rs165599 on catechol-O-methyltransferase (COMT) gene by PCR-RFLP. Methods: DNA samples of rs165599 known genotypes by sequencing, using techniques including PCR amplification, restriction fragment length polymorphism, and agarose gel electrophoresis to determine parameters for optimization of the protocol.

APPLICATION OF PCR-RFLP TECHINIQUE IN GENOTYPING

FOR RS165599 POLYMORPHISM ON COMT GENE

Dinh Viet Hung 1 ; Dang Tien Truong 1 ; Cao Tien Duc 2 ; Tran Hai Anh 1

SUMMARY

Objectives: To establish and complete a protocol for genotyping of single polymorphism rs165599 on catechol-O-methyltransferase (COMT) gene by PCR-RFLP Methods: DNA samples of rs165599 known genotypes by sequencing; using techniques including PCR amplification, restriction fragment length polymorphism, and agarose gel electrophoresis to determine parameters for optimization of the protocol Results: Determining the temperature at 56 o C and 33 cycles were optimal for the protocol to genotype rs165599/COMT gene Conclusion: The genotyping protocol for rs165599 polymorphism was established, which would be able to apply in elucidating the association of this polymorphism in neuropsychological disorders

* Keywords: COMT gene; PCR-RFLP; rs165599

INTRODUCTION

COMT gene (Catechol-O-methyltransferase)

encodes COMT enzyme, which several

enzymes involved in degeneration of

catecholamine neurotransmitters (including

dopamine, epinephrine, and norepinephrine)

COMT exists in two different forms:

soluble short form, found in cell cytoplasm

(S-COMT), and a larger form binding to

the cell membrane (MB-COMT) Lachman

et al (1996) found a single nucleotide

polymorphism (SNP) on the COMT gene

that encodes enzymes to change the

activity of the COMT enzyme three to four

times [6] COMT is the most studied gene

in behavioral genetics since the first

report of the American Association of

Schizophrenia in 1996 [7] This gene has

been studied extensively due to many

factors, including the area associated with schizophrenia on chromosome

22 containing a significant 22q11 deletion, relating to metabolism of catecholamine - neurotransmitters regarded as having an association with mental disorders and psychiatric treatment [3] Since then, many independent case-control studies, family-based as well as genome-wide studies have found associations of

polymorphism on COMT gene with mental disorders [2, 3, 8] Studies on COMT

gene has also showed interactions with environmental risk factors related to mental disorders such as marijuana [4, 5] The most common polymorphisms of

COMT gene are rs4680, rs7378645,

and rs165599 Rs4680 is the most studied, but found no association with schizophrenia in a Vietnamese population [1]

1 Vietnam Military Medical University

2 103 Military Hospital

Corresponding author: Tran Hai Anh (anhhtr@yahoo.com)

Date received: 10/04/2019

Date accepted: 20/05/2019

Another polymorphism has attracted

attention is rs165599, which is located at

the beginning of 3' end of the COMT

gene, next to exon 6 This study aims at:

Building a PCR-RFLP (restriction fragment

length polymorphism) protocol, identifying

genotypes of polymorphic rs165599/COMT

for studies that evaluate the association of

this SNP in schizophrenia

MATERIALS AND METHODS

1 Materials

Three DNA samples with known

genotypes for polymorphic rs165599 on

COMT gene by sequencing method

These genotypes were AA, GG,

and AG, respectively Other molecular

chemicals include: forward and reverse

primers (5’-CGACTTAGTACATCCTTC-3’

and 5’-GAGTAGAATCTTGGCTAG-3’,

respectively) were synthesized by Phusa

Biochem; Hot-tag master mix (QIAGEN),

Emzym MspI (Invitrogen), Agarose (Thermo),

pure water (Invitrogen), TBE 1X

2 Equipment

Equipment used in PCR: small shaker

for mixing - MS3 digital (IKA, USA);

centrifugal centrifuge - E-centrifuge (Wealtec,

USA); PCR machine - Proflex (ABI, USA)

Equipment used in electrophoresis: electronic

scales - TE612 (Sartorius, Germany);

microwave oven (Sharp, Japan);

electrophoresis (Scie-plas, UK); gel

camera (UK) and some basic molecular

equipment

3 Methods

* PCR amplification:

Amplification of COMT gene segment

containing rs165599 by PCR reaction

20 µL PCR reaction includes 100 ng genomic DNA, 0.5 µM per primer, Master mix i-taq 1X, and sufficient water The amplification reaction runs on the ProFlex PCR system thermal cycle as follows: 94°C for 2 mins; 33 cycles of 94°C for 20 s, 56°C for 10 s, 72°C for 40 s; and the last step was 72°C in 4 mins; stored at 4°C PCR products with calculated size are theoretically 628 bp

* Enzyme treatment:

PCR products were treated with MspI enzyme according to the manufacturer's recommendations, summarized as follows:

10 µL PCR products, 0.5 µL enzyme, 2.5 µL Buffer, and 12 µL water The mixture was incubated at 37°C for 5 mins Enzyme MSPI will cut the CC/GG sequence of section 628 bp, forming two segments with sizes of 403 bp and 225 bp, respectively

* Agarose gene electrophoresis:

PCR products after being treated with MSPI enzyme, ran on 2% agarose electrophoresis, at 100 V, for 60 minutes Next, took the gel and determined the results Results of electrophoresis of

samples with AA genes showed one band

sized 628 bp; samples with AG genotype showed three bands sized 628 bp, 403 bp, and 225 bp, respectively; and samples with GG genotypes showed two bands sized 403 bp and 225 bp, respectively

RESULTS AND DISCUSSION

1 Optimization of PCR

Many factors affect the results of PCR

reactions such as annealing temperature

(Ta), annealing time, time of the extension,

enzyme activity, dNTP concentration,

Mg++ concentration Optimizing the PCR

reaction needs to optimize all those factors

to achieve the best results However,

this work consumes much time, efforts,

and chemicals Therefore, the actual

optimization of PCR reaction usually performs the selection of standard reaction components and optimizes the annealing time (Ta) first In the present study, we used 2x master mix solution of INtRON to save time and cost optimization of factors related to the reaction component Ta of the reaction depends on the melting temperature (Tm) of the primer and usually less than

5 - 10oC We conducted optimization tests

at Ta range of 55 - 65oC (figure 1)

Figure 1: Results of optimization of Ta at 55oC, 60oC, and 65oC

(55, 60, 65 were product bands of PCR reaction at T a as 55 o C, 60 o C, and 65 o C, respectively; (-): negative control (in 60 o C); M: Marker 100 bp)

The results in figure 1 showed that the reaction occurred at Ta of 60°C for the darkest signal band (no bands at the other temperatures), with no by-products The Ta was close to the theoretical calculations, and was no contaminated products

It can be showed that the optimal Ta for primer pairs ranged about 55°C to 60°C, therefore, we continued testing the optimization reaction at three Ta of 55°C, 56°C,

and 58°C (figure 2)

Figure 2: Results of optimization of Ta at 55oC, 56oC, and 58oC

(55, 56, 58 were product bands of reaction at at T a as 55 o C, 56 o C, and 58 o C; (-): Negative control; M: Marker 100 bp)

The results in figure 2 showed that the reaction occurred at a Ta of 56°C giving the strongest band, then the product at Ta of 55°C, without by-product The Ta was closed

to the theoretical calculation, no contaminated products It can be concluded that the optimal Ta for the primer pairs was about 56oC

After repeatedly adjusting various conditions for the PCR reaction, we concluded that the annealing temperature of the reaction was 56°C and the number of cycles was 33 were optimal Other components were recommended by the manufacturer

Thus, we have successfully optimized the PCR of the COMT fragment containing

polymorphic rs165599

2 Results of genotyping determined by RFLP

PCR products were treated with MspI enzymes according to the manufacturer's recommendations The results were shown in figure 3 below

Figure 3: Electrophoresis results after enzyme treatment

(AG, GG, AA were product bands with corresponding genotypes of rs165599, respectively; (-): Negative proof; M: Marker 100bp)

The results of enzyme treatment in samples with corresponding genotypes showed that the products were in accordance with those of theoretical calculations Sample with AG genotype had three bands as 628, 403, and 225 bp; sample with GG genotype had two bands as 403 bp and 225 bp; and the sample with AA genotype had only one

628 bp band

Thus, we had successfully established the protocol for identifying the genotypes of

polymorphic rs165599 on COMT gene This protocol is simple, inexpensive, not requiring

complex and expensive equipment Thus, this protocol is applicable in studies elucidating the association of this polymorphism with neuropsychiatric diseases with a large

sample size and less expense

(-) AG GG AA M

628

403

225

CONCLUSION

Successfully developed the protocol

for genotyping polymorphic rs165599 on

COMT gene, serving studies on the

association of the polymorphism and

schizophrenia

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