Isolation Of Dna From Fish Sperm

Isolation Of Dna From Fish Sperm
Does anyone have a protocol for DNA/RNA extraction from fish mature sperms and oocytes?
Hello, I need to extract DNA and RNA from fish mature spermatozoa and unfertilised oocytes. I am also looking for a protocol to isolate mature spermatozoa from fish semen/testis and any advice on how to proceed with oocyte DNA/RNA extraction.
Hellenic Centre for Marine Research
Here at our institute we have extracted DNA from eggs for paternity evaluations, and RNA for gene expression. The persons that can help you with the protocol are
Dr Costas Tsigenopoulos and Dr Elena Sarropoulou.
Please contact me on my email ( mylonas@hcmr.gr ) to make the connection with the above researchers.
I have attached my methods for DNA and RNA extraction from lymphocytes, which of course are diploid cells. It may require some adaptions but basically the principles should be similar, as you basically need to lyse the appropriate membranes (cell and nucleic) to isolate different parts of the cell. The methods are based on the fact that DNA is confined to the nucleus and RNA is found in the cytosol. The nuclei of the lymphocytes extracted from peripheral blood remain in tact. If this is the same for sperm and oocytes then the same method can be used but may need adaptions for haploid cells. The RNA isolation from the cytosol is very similar to any other nucleotide extraction.
Extraction of DNA and RNA from lymph
We regularly genotype fish sperm. We release it, spin it down gently, and from there we treat it just like a piece of tissue with a typical SDS genomic DNA extraction buffer and a normal phenol:chloroform extraction. Let me know if you want details!
Oocytes obviously have far less DNA, I doubt the same method would work there.
We simultaneously extract DNA from fish fins, muscle, and eggs using the CTAB protocol or the Qiagen DNEasy blood and tissue kit, and get good results for the first two, while the DNA from eggs is smeary and has very low quality readings. Does anyone know what's in the eggs that is causing this, and have you come across an extraction procedure that significantly improves the situation? (by the way, the eggs were preserved in ethanol, RNAlater and Allprotect reagent, but the genomic DNA is consistently poor).
Does anyone have an RNA extraction protocol for fish/salmonid sperm?
I'm looking to extract high quality RNA from Atlantic salmon sperm and am having difficulty finding a protocol that isn't mammal/human specific.
Protocol for High Quality DNA Isolation from Mouse Oocytes?
I am looking for a protocol optimized for the DNA (RNA-free) isolation of mammalian (preferentially mouse) MII oocytes.
I have different pools of 20-80 oocytes and would like to proceed with DNA-specific analysis.
I performed some bibliographic review and I see every different paper with a different protocol, so I thought it would be useful to ear someone with experience in this particular protocol.
Which procedure or kit is best for DNA and RNA extraction from few sperm cells ?
1. What is best procedure or kit for DNA and RNA extraction from few sperm cells in your work experience? We will sort sperm cells in process so we have to extract both RNA and DNA from few sperm cells. which kit or procedure is best?
2. What is the best way to keep sperm cells safe for while ( like weeks) before extracting DNA and RNA from them?
What's the best way to make an RNAse-free solution?
I need to make a 10 mM Tris solution, pH 7.4, that is RNAse free. I have RNAse-free Tris and RNAse free water to make the 10 mM Tris solution. How's the best way logistically to weigh out the amount needed (~12 mg) on an analytical balance in a way that doesn't contaminate the solution with RNAse. Typically I would use an autoclaved spatula and weigh paper, but I'm pretty sure neither of those in our lab are RNAse free. Any tips would be welcome!
Anyone have a good protocol for extracting DNA from Fish eggs?
Low levels of DNA in early develkopmental stages of fish eggs. DNAEASy kits etc dont appear to work well. Chelex protocol is Ok anyone have ideas on DNA prepcipitation protocols we could try?
Can low 260/230 ratio in RNA extraction interfere with qPCR or RT-PCR result?
I extracted human embryoid body RNA for PCR analysis using Trizol reagent. RNA conc. is between 50-200 ng/ul, and 260/280 ratio is about 1.7-2.1,so these are really good, but 260/230 ratio is extremely low ~0.3-0.7.
The first time I used GeneJET RNA Purification Kit (ThermoSci), both RNA conc. and 260/230 ratio are lower than when I switch to use Trizol reagent. Can I use these RNA for PCR analysis?
How to avoid Primer - Dimer Formation and get our gene amplified ?
I have to amplify a gene from C. elegans genomic DNA and I have designed the primers. But after amplification with PCR and running on gel I see the band way down the ladder, that is, the band is below the lowest band on the ladder of 250 bps.
I have also tried the Temperature GRADIENT method for PCR but it doesn't help.
What is the critical step in DNA extraction of fish sperm from a long term cryopreservation? Is there any suitable method to extract these samples?
I had frozen sperm from Oreochromis mortimeri prepared in 1990, and the template is also limited.
There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of so...
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Method for the extraction of genomic DNA from fish blood or sperm

A simple and efficient process for the extraction of genomic DNA from a fish, requiring a minuscule amount of blood or sperm from the fish without sacrificing it, comprises: adding blood or semen taken from the fish into a first buffer for cell-lysis to create a first suspension; centrifuging the first suspension to collect a precipitated nuclear pellet; adding the precipitated pellet into a second buffer for nuclear-lysis to create a second suspension; and extracting DNA from the second suspension.

C07H1/00 ; C07H21/04 ; (IPC1-7): C07H1/00; C07H21/04

Brem et al., Aquaculture, 68: 209-219, 1988.
Dunham et al., Trans. Am. Fish Soc., 116: 87-91, 1987.
Ivics et al., Mol. Marine Biol. Biotechnol., 2: 162-173, 1993.
Khoo et al., Aquaculture, 107: 1-19, 1992.
Penman et al., Aquaculture, 85: 35-50, 1990.
Penman et al., Mol. Repr. Dev., 30: 201-206, 1991.
Zhang et al., Mol. Repr. Dev., 25: 3-13, 1990.
Taggart et al., J. Fish Biol. 40: 963-965, 1992.
Cummings et al., Biotechniques, 17(3): 426-430, 1994.

(a) adding blood or semen taken from the fish into a first buffer for cell-lysis to create a first suspension;
(b) centrifuging the first suspension to collect a precipitated pellet;
(c) adding the precipitated pellet into a second buffer containing no proteinase for nuclear-lysis to create a second suspension; and
(d) extracting DNA from the second suspension with an organic solvent.
(i) adding an equal amount of Tris-saturated phenol(pH 8.0) to the second suspension and centrifuging the resulting mixture to obtain a first supernatant;
(ii) adding an equal amount of phenol:chloroform mixture(1:1(v/v)) to the first supernatant and centrifuging the resulting mixture to obtain a second supernatant;
(iii) adding an equal amount of isopropanol to the second supernatant to precipitate DNA; and
(iv) recovering the precipitated DNA.
The present invention relates to a process for the extraction of genomic DNA from fish blood or sperm; and, more particularly, it pertains to a simple and efficient process for the extraction of genomic DNA that requires a limited amount of blood or sperm from fish without sacrificing it.
Recent progress made in the studies on piscine genes has developed various techniques for the extraction of genomic DNA from fish.
One of such techniques is to extract DNA from fish liver, fin or muscle, or even the whole body of small fish, which is based on the technique developed for mammals(see Brem, G. B., et al., Aquaculture, 68, 209-219(1988); Dunham, R. A., et al., Trans. Am. Fish Soc., 116, 87-91(1987); Ivics, Z., et al., Mol. Marine Biol. Biotechnol., 2, 162-173(1993); Khoo, H. W., et al., Aquaculture, 107, 1-19(1992); Penman, D. J., et al., Aquaculture, 85, 35-50(1990); Penman, D. J., et al., Mol. Repr. Dev., 30, 201-206(1991); and Zhang, P., et al., Mol. Repr. Dev., 25, 3-13(1990)). Another process developed for extracting DNA from salmonid fish is also based on the technique for mammals(see Taggart, J. B., et al., J. Fish Biol., 40, 963-965(1992)). However, these techniques are very complicated and require expensive proteinases, RNases, thermostatic apparatus, and a great deal of labor. Therefore, the analysis of large number of samples may be tiresome, laborious, and not economical in many situations. Further, fish is inevitably sacrificed or injured seriously in case of these methods.
A method for extracting genomic DNA from fish blood or sperm has been recently developed for the purpose of DNA fingerprinting(Cummings, S. A. and G. H. Thorgaard, Biotechniques, 17(3), 426-430(1994)). In this process, blood is obtained from the heart of an anesthetized salmon by using a syringe containing 1×SSC(0.15M NaCl, 0.015M sodium citrate) and the blood sample so obtained is put into a tube. A large amount of distilled water is added to the tube to disrupt the cells, and then, 5×SSC is added to make the solution isotonic. The mixture is centrifuged to obtain precipitate, which is washed with NaCl and EDTA, and then centrifuged again to obtain precipitate. A buffer solution containing proteinase K is added to the precipitate and the mixture is stirred at 60° C. overnight to digest the precipitate. The mixture is cooled to a room temperature and ethanol is added to precipitate DNA, which is then separated and washed with ethanol. The obtained DNA is dried and dissolved in 1×TE buffer at 37° C. overnight. In case of sperm, a buffer solution containing proteinase K is added and the mixture is maintained at 60° C. overnight to digest the sperm; and then the same procedures as in the case of blood are repeated.
However, in this process, expensive proteinase K is employed for the extraction of DNA, which in turn requires a thermostatic apparatus and a stirrer. Further, it takes a long time to complete the process. More importantly, a considerable amount of proteins may precipitate together with DNA during the process.
Therefore, efforts have continued for the development of a simple and economical process for the extraction of genomic DNA from fish.
Accordingly, it is a primary object of the present invention to provide a simple and economical process for the extraction of genomic DNA having a high purity from fish without sacrificing the latter.
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the result of agarose gel electrophoresis for verifying the purity of the DNA samples extracted from nile tilapia(Oreochromis niloticus) and mud loach(Misgurnus mizolepis) in accordance with the inventive process; and
FIG. 2 represents the result of agarose gel electrophoresis by using the DNA samples extracted from various fish species in accordance with the process of the present invention and the DNA sample extracted from blood of nile tilapia using the process of Cummings and Thogaard, supra.
DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated in their entirety by reference.
The process of the present invention requires only a small amount of blood or semen from fishes, for example, 5 to 10 μl of blood or 1 to 2 μl of semen. The blood or semen sample is added to a first buffer for cell-lysis to create a first suspension.
The first buffer for cell-lysis consists essentially of a surfactant for lysing a cell membrane, for example, Triton X-100 in a concentration ranging from 1 to 5%, preferably, 2.5%. An exemplary buffer may consist of 12.5 mM citric acid, 25 mM sodium citrate, 41 mM dextrose, and 2.5% Triton X-100. The buffer, owing to the characteristic of the surfactant therein, disrupts only the cell membrane without disrupting nuclear membrane, which may be completed within 2 or 3 seconds only.
After disrupting the cell membrane, the first suspension is centrifuged at a gravity ranging from 1,800 to 2,000 g for a time period ranging from 5 to 10 minutes to precipitate the nuclear pellet. The nuclear pellet collected is resuspended using a gentle vortexing, and a second buffer for nuclearlysis is added to create the second suspension.
The second buffer for nuclear-lysis preferably comprises sodium dodecyl sulfate(SDS) in a concentration ranging from 0.1 to 1.0%, preferably, 0.5%. A representative buffer may consist of 10 mM Tris-Cl, 1 mM EDTA, 0.5 M NaCl, and 0.5% SDS. pH of the buffer ranges from 6.8 to 8.5, preferably, 8.0. The reaction of the buffer to the nuclear pellet may be completed within 5 to 10 seconds.
The above procedures do not require any particular temperature control, and can be carried out at a room temperature without restriction.
After the lysis of the nuclear membrane, the second suspension is extracted using organic solvent and the organic layer is centrifuged to obtain a supernatant containing DNA. An exemplary organic solvent which may be used for the extraction is Tris-saturated phenol(pH 8.0). The supernatant is further treated with another organic solvent, e.g., phenol:chloroform mixture(1:1(v/v)), in accordance with the same procedure as above to obtain a supernatant. To the supernatant is added isopropanol to precipitate DNA, and then the precipitated DNA is recovered from the solution and dissolved in 1×TE buffer for a direct use in the genetic analysis.
The buffer for the cell-lysis or nuclear-lysis used in the present invention is stable at a room temperature and has long-term storage life. Further, since the present invention employs only the collected nuclei for the DNA extraction and not the whole cell, it is not required to remove a large amount of proteins; and, therefore, a simple extraction will be sufficient to remove residual proteins. In addition, unlike the prior art processes, expensive RNase is not needed since the removal of cellular RNA, which is usually present in the cytoplasm, is unnecessary. The present invention employs isopropanol which is much more effective than ethanol in precipitating DNA, and, therefore, relatively small amount thereof is sufficient to precipitate DNA in the supernatant even at a room temperature.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Ten μl of blood(1.5×10 7 cells) was collected from a nile tilapia(Oreochromis niloticus) by using a capillary or disposable syringe. 1,200 μl of buffer for cell-lysis(12.5 mM citric acid, 25 mM sodium citrate, 41 mM dextrose, 2.5% Triton X-100) was added to the blood, and mixed thoroughly for 5 seconds. The mixture was microcentrifuged at 2,500 rpm for 10 minutes to collect nuclear pellet.
The collected nuclear pellet was subjected to a gentle vortexing, and 600 μl of a buffer for nuclear-lysis(10 mM Tris-Cl, 1 mM EDTA, 0.5 M NaCl, 0.5% SDS, pH 8.0) was added. The suspension was mixed slowly for 10 seconds. To the mixture was added an equal amount of Tris-saturated phenol(pH 8.0), and the resultant was mixed for 10 seconds and then centrifuged at 14,000 rpm for 3 minutes to obtain a supernatant. An equal amount of phenol:chloroform mixture(1:1(v/v)) was added to the supernatant and the above procedures were repeated to obtain a supernatant.
To the supernatant was added an equal amount of isopropanol to precipitate DNA. The precipitated DNA is recovered from the solution and dissolved in 100 μl of 1×TE buffer(10 mM Tris, 1 mM EDTA, pH 8.0).
The O.D.(optical density) value of the resulting solution was measured at 260 nm and 280 nm by using a UV-Vis spectrophotometer. The amount of DNA in the solution can be calculated from the O.D. value at 260 nm(OD 260 ), in accordance with the method described by Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd Ed., 3, Appendix E.5, wherein it is described that an OD 260nm of 1.0 corresponds to approximately 50 μg/ml for double-stranded DNA. As the OD 260 value of the resulting solution was measured as 6.01, the concentration of DNA therein was calculated as about 301 μg/ml, and the amount of DNA in 100 μl of the solution was determined as 30.1 μg. Considering the genome size of nile tilapia(2.6 pg of DNA/cell; Brem, et al., Aquaculture, 68, 209-219(1988)) and the number of cells used in the experiment, the yield of DNA was calculated as about 77%.
Further, the purity of the extracted DNA was determined from the OD 280 value of the DNA solution, in accordance with the method described by Sambrook et al., supra, wherein it is described that pure DNA has an OD 260 /OD 280 value of 1.8. As the OD 280 value of the resulting DNA solution was measured as 3.31, the OD 260 /OD 280 value was calculated therefrom as 1.81 and, therefore, it was confirmed that the extracted DNA was pure.
The same procedures as in Example 1 were repeated by using 1 μl of semen(3×10 7 cells) from nile tilapia instead of 10 μl of blood. The yield of DNA was also about 75% and the extracted DNA was pure.
For the purpose of confirming the purity of DNA obtained in Example 1, an agarose gel electrophoresis was carried out as follows.
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Isolation Of Dna From Fish Sperm

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