Evaluation of eight protocols for genomic DNA extraction of <i>Hypostomus commersoni</i> Valenciennes, 1836 (Loricariidae: Siluriformes)

Mezzomo, P.; Mielniczki-Pereira, A. A.; Sausen, T. L.; Marinho, J. R.; Cansian, R. L.

doi:10.1590/1519-6984.229278

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Brazilian Journal of Biology

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Original Article • Braz. J. Biol. 81 (3) • Jul-Sep 2021 • https://doi.org/10.1590/1519-6984.229278 copy

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml^-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Keywords:
DNA extraction; PCR-RAPD; eye tissue; Proteinase K; suckermouth catfish

Variables	Efficient	Indifferent	Inefficient
Specimens conservation	Fresh tissues (approx. 27 °C)	-	Tissues in 70% and 95% ethanol at 27 ºC
Specimens conservation	Fresh tissues (approx. 27 °C)	-	Tissues at -10 ºC and -20 ºC
Maceration	Chemical lysis	Lysis with LN₂	Mechanical lysis
Maceration	Chemical lysis	Lysis with LN₂	Manual lysis
Target-Tissue	Ocular tissue	–	Caudal fin
			Dorsal fin
			Muscle
			Gills
			Blood
Time of incubation	1h (55 ºC)	–	–
Time of incubation	12h, 24h, 36h (27 ºC)	–	–
RNAse	Absence	Presence	–
Proteinase K	Presence in high concentration (20 mg.ml^-1)	–	–
Storage after extraction	-10 ºC -20 ºC	–	4 ºC
Quantification	In the day of the extraction	–	–
Quantification	After 10, 20 and 30 days of storage	–	–

Reagents
STE Buffer	0.1 M NaCl + 10 mM Tris + 1 mM EDTA (pH 8.0)
Lysis Buffer	2.5 mM Tris-HCl pH 8.0 + 0.1 mM EDTA pH 8.0 + 0.1% SDS + 8.0 mM NaCl
Proteinase K	20 mg.mL^-1
CIA	Chloroform + isoamylic alcohol (1:24 v/v)
TE Buffer	10 mM Tris pH 8.0 + 1 mM EDTA pH 8.0
TBE Buffer	500 mM Tris-HCl + 60 mM boric acid + 83 mM EDTA
Steps of extraction
1. Collect 20 mg of fresh tissue.
2. Wash in 500 µl of STE (cold) and discard.
3. Add 500 µl of lysis buffer, 2 µL of β-Mercaptoetanol and 13 µl of proteinase K, in this order, and incubate at 55 ºC for 1 hour (alternatives include the incubation for 12, 24 or 36 hours at 27 ºC).
4. Add 700 µl of CIA and homogenize by inversion.
5. Centrifuge for 10 minutes at 9,000 rpm and transfer the supernatant to a new tube.
6. Repeat steps 4 and 5 (3x).
7. Transfer the aqueous phase to a new tube containing 900 µl of absolute isopropanol (cold), homogenize by inversion.
8. Incubate for 2 hours at –20 ºC.
9. Centrifuge for 15 minutes at 9,000 rpm and discard the supernatant.
*10. Wash the resulting pellet* with ethanol analytical grade (cold).**
11. Discard all the volume and incubate at 37 ºC for 15 minutes.
12. Add 100 µL of TE buffer and incubate for 1 hour at approximately 27 ºC.
13. Storage at – 10 ºC until use.

Method (Protocol)	CF	DF		GL	BD	OT	Methods mean (ng/µl)
	Total DNA amount (ng/µl)
	Mean ± SD
1. Doyle and Doyle (1987)DOYLE, J.J. and DOYLE, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, vol. 19, pp. 11-15. http://dx.doi.org/10.12691/jfnr-2-7-6. http://dx.doi.org/10.12691/jfnr-2-7-6...	120±21.2	139±26.9	198±10.6	110±22	365±60.4	400±26.8	222
2. Medrano et al. (1990)MEDRANO, J.F., AASEN, E. and SHARROW, L., 1990. DNA extraction from nucleated red blood cells. Biotechnology, vol. 8, no. 1, pp. 43. PMid:2182076.	175±8.6	114±23.7	155±32.8	137±27.8	375±115.2	520±62.8	246
3. Whitmore et al. (1992)WHITMORE, D.H., THAI, T.H. and CRAFT, M., 1992. Gene Amplification Permits Minimally Invasive Analysis of Fish Mitochondrial DNA. Transactions of the American Fisheries Society, vol. 12, no. 2, pp. 170-177. http://dx.doi.org/10.1577/1548-8659(1992)121<0170:GAPMIA>2.3.CO;2. http://dx.doi.org/10.1577/1548-8659(1992...	116±18.0	74±19	173±35.3	125±6.5	335±70.3	455±48.8	216
4. Aljanabi and Martinez (1997)ALJANABI, S.M. and MARTINEZ, I., 1997. Universal and rapid salt-extraction of high-quality genomic DNA for PCR-based techniques. Nucleic Acids Research, vol. 25, no. 22, pp. 4692-4693. http://dx.doi.org/10.1093/nar/25.22.4692. PMid:9358185. http://dx.doi.org/10.1093/nar/25.22.4692...	181±17.3	90±12.3	120±33.2	110±12.6	455±60.1	465±50.9	237
5. Faleiro et al. (2003)FALEIRO, F.G., FALEIRO, A.S.G., CORDEIRO, M.C.R. and KARIA, C.T., 2003. Metodologia para operacionalizar a extração de DNA de espécies nativas do cerrado. Planaltina. Embrapa Cerrados, vol. 1, pp. 1-6.	168±39.9	150±24.5	205±28.1	155±10.6	425±20.4	505±63.9	268
6. Barrero et al. (2008)BARRERO, N.M.L., POVH, J.A., RIBEIRO, R.P., GOMES, P.C., JACOMETO, C.B. and LOPES, T.S., 2008. Comparison of DNA extraction protocols of fish fin and larvae samples: modified salt (NaCl) extraction. Ciencia e Investigación Agraria, vol. 35, pp. 65-74.	157±29.0	97±9.6	207±32.6	175±18.7	460±51.9	555±47.3	275
7. Coppini (2009)COPPINI, V.J., 2009. Variabilidade genética em Oligoryzomys nigripes através de marcadores RAPD e cariotipagem, tendo o rio Uruguai como barreira geográfica. Erechim: Programa de Pós-Graduação em Ecologia, Universidade Regional Integrada do Alto Uruguai e das Missões, Dissertação de Mestrado. [[Q7: Q7]]	186±6.6	160±44.4	170±19.1	135±1.6	330±17.1	516±22.2	250
8. Improved protocol (2020)	209±15.7	220±33.5	216±0.8	210±4.1	463±9.7	712±18.8	438
Tissues Mean	164	131	180	145	404	516	-

Method (Protocol)	CF	DF	MC	GL	BD	OT
	Absorbance ratio (OD 280/260 nm)						Methods Mean
	Mean ± SD						Methods Mean
1, Doyle and Doyle (1987)DOYLE, J.J. and DOYLE, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, vol. 19, pp. 11-15. http://dx.doi.org/10.12691/jfnr-2-7-6. http://dx.doi.org/10.12691/jfnr-2-7-6...	1.5 ±0.016	0.8 ±0.08	1.4 ±0.08	1 ±0	1.9 ±0.08	1.8 ±0.12	1.4
2, Medrano et al. (1990)MEDRANO, J.F., AASEN, E. and SHARROW, L., 1990. DNA extraction from nucleated red blood cells. Biotechnology, vol. 8, no. 1, pp. 43. PMid:2182076.	1.8 ±0.08	0.6 ±0.08	1.6 ±0.08	1 ±0.08	1.4 ±0.08	1.6 ±0.08	1.3
3, Whitmore et al. (1992)WHITMORE, D.H., THAI, T.H. and CRAFT, M., 1992. Gene Amplification Permits Minimally Invasive Analysis of Fish Mitochondrial DNA. Transactions of the American Fisheries Society, vol. 12, no. 2, pp. 170-177. http://dx.doi.org/10.1577/1548-8659(1992)121<0170:GAPMIA>2.3.CO;2. http://dx.doi.org/10.1577/1548-8659(1992...	1 ±0.14	0.7 ±0.08	1.6 ±0.08	1.2 ±0	1.6 ±0.08	1.8 ±0.08	1.3
4, Aljanabi and Martinez (1997)ALJANABI, S.M. and MARTINEZ, I., 1997. Universal and rapid salt-extraction of high-quality genomic DNA for PCR-based techniques. Nucleic Acids Research, vol. 25, no. 22, pp. 4692-4693. http://dx.doi.org/10.1093/nar/25.22.4692. PMid:9358185. http://dx.doi.org/10.1093/nar/25.22.4692...	1.1 ±0.16	0.8 ±0.08	1 ±0.08	1 ±0	1.7 ±0.08	1.6 ±0.08	1.2
5, Faleiro et al. (2003)FALEIRO, F.G., FALEIRO, A.S.G., CORDEIRO, M.C.R. and KARIA, C.T., 2003. Metodologia para operacionalizar a extração de DNA de espécies nativas do cerrado. Planaltina. Embrapa Cerrados, vol. 1, pp. 1-6.	1.1 ±0.08	0.9 ±0.08	1.6 ±0.08	1.1 ±0.14	1.6 ±0	1.9 ±0.08	1.4
6, Barrero et al. (2008)BARRERO, N.M.L., POVH, J.A., RIBEIRO, R.P., GOMES, P.C., JACOMETO, C.B. and LOPES, T.S., 2008. Comparison of DNA extraction protocols of fish fin and larvae samples: modified salt (NaCl) extraction. Ciencia e Investigación Agraria, vol. 35, pp. 65-74.	1.1 ±0.08	0.9 ±0.08	1.3 ±0.08	1.2 ±0	1.8 ±0.08	1.7 ±0.08	1.3
7, Coppini (2009)COPPINI, V.J., 2009. Variabilidade genética em Oligoryzomys nigripes através de marcadores RAPD e cariotipagem, tendo o rio Uruguai como barreira geográfica. Erechim: Programa de Pós-Graduação em Ecologia, Universidade Regional Integrada do Alto Uruguai e das Missões, Dissertação de Mestrado.	1.5 ±0.08	1.1 ±0.12	0.8 ±0.08	1.1 ±0.08	1.6 ±0	1.8 ±0.08	1.3
8, Improved protocol (2019)	2 ±0	1.5 ±0.08	1.7 ±0.08	1.5 ±0.08	1.9 ±0.08	2 ±0.08	1.8
Tissues Mean	1.4	0.9	1.3	1.1	1.7	1.8	-

Primer	Sequence 5’ - 3’	Score of Amplified DNA bands
Primer	Sequence 5’ - 3’	40 ng/µl	20 ng/µl	10 ng/µl
OPW-01	CTCAGTGTCC	6	6	6
OPW-02	ACCCCGCCAA	6	8	8
OPW-03	GTCCGGAGTG	4	8	7
OPW-04	CAGAAGCGGA	7	9	7
OPW-05	GGCGGATAAG	4	4	-
OPW-06	AGGCCCGATG	7	8	-
OPW-07	CTGGACGTCA	7	8	-
OPW-08	GACTGCCTCT	5	5	-
OPW-09	GTGACCGAGT	6	6	-

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