Introduction
Colony hybridization is an application of nucleic acid hybridization that is combined with conventional environmental microbiological sampling and viable planting procedures (1). This process utilizes a gene probes, which is radio actively marker, which attaches to complimentary base pairs from a single strand of bacteria DNA. This has proven to be a useful procedure for efficiently screening bacteria clones by RNA-DNA or DNA-DNA hybridization (4). This technique is accomplished by growing microbial communities from samples collecting at the research site on an agar plate, then transferring the colonies to filter paper to be lysed and denatured. After this step a labeled probe is added to bind to complementary strand of DNA and this product is assessed for any hybridization which is visualized as a black spot when looked at under x-ray. The original protocol was developed by Grunstein and Hogness (2).
Use of technology
This technique can be used to find similar microbial communities in a sample. If there is a known gene or sequence nucleic acids a researcher is looking for in a particular samples, whether it is taken from soil, water, or the air. This can also search for particular plasmids, which is extra chromosomal DNA (3). This technique allows a researcher to pick a single gene from the whole genome of a bacterium of interest, while at the same time saving the researcher from having to run DNA isolation assay or a PCR assay. The con’s of colony hybridization is that this assay is very time consuming, with all of the incubation this research takes days to complete. Another pitfall of this technique is that is only applies to microbes that can withstand the plating process, meaning 99% of the microbe species in the sample with not be incorporated into this experiment.
Materials and Protocol
1. Isolate and grow cultures in a suitable nutrient medium (5). Bacteria can be placed on selective agar for microbe colonies that are sensitive to plating; selective plates are used to facilitate growth of a particular microbe while inhibiting the growth of other microbes.
2. Transfer a sample of the colonies onto a solid matrix such as a nitrocellulose or nylon membrane (5). This procedure is done after the colonies have grown on the plate after incubation, which is usually done over night at 32oC however time and temperature can change depending upon the bacteria being assayed. The filter, which can be either Nylon or nitrocellulose filter, is placed on top of the bacterial colonies on the agar plate and incubated once the colonies have transferred to the filter for 24 hours at 30oC.
3. The cells on the membrane are lysed and the DNA is then denatured (5). After incubation the filters were placed and incubated on a series of saturated papers that lyses the cell and denatures the DNA and helps it absorb into the filter paper. The first saturated paper contained 1M sorbitol, 40 mM EDTA with a PH of 8.0, and 50 mM DTT, this was incubated for 15 min at 30° C; the second saturated paper contans 1 M sorbitol, 40 mM EDTA with a PH of 8.0, and 10% glusulase, and incubated for 3-4 h, at 37° C; the third saturated paper contained 0.5 M NaOH, and 10x SSC, and was incubated for 8 min at 37° C; the fourth and fifth saturated papers contained 0.5 M Tris-HCl (pH 7.4), 10x SSC, and were both incubated for 4 min at 37° C (5).The filters were then transferred to a filtration block and, with suction applied, 200 ml of 3x SSC and 100 ml of chloroform were poured through the filter, and then the filters are baked in a oven at 80° C for 2 h(5).
4. A labeled gene probe is added to the matrix and hybridization takes place (5). The gene probe is made by isolating a piece of genetic information and the label is usually either 32P- or biotin. The hybridization occurs when the bases of the DNA match the sequence of the gene probe. When a gene probe finds its complementary DNA sequence they bind together and the specific genetic properties can be detected. This process begins with soaking the baked filter in 3x SSC for 15 minutes followed by 2 hours of incubation in the gene probe solution. Once the 2 hours incubation is completed the filter is then taken out and allowed to incubate at 65oC for 24 while the hybridizations are occurring (4).
5. The matrix is rinsed to remove the non-hybridized probe molecules (5). The wash reduces the level of background noise of the probe. The filter was washed with 3x SSC, then 2x SSC and lastly 1x SSC.
6. For radioactive probes, one uses autoradiography and the matrix is placed on x-ray film (5). This is when the colonies with the gene probe hybridization are visualized. If the colony has had a hybridizing event the bacteria will appear black under the X-ray autoradiograph.
7. Compare the x-ray film with the master plate to see which colonies had probe hybridization. These are the colonies that contained the specific sequence that actually hybridized with the probe (5). If everything is done properly it should be an easy task to compare the developed autoradiograph to the original plated bacterial colonies and to see which colony has had hybridization events.
Actual DNA probe results; Black dots showing colonies with gene probe hybridization
Work Cited
1. Atlas, Ronald M. Microbial ecology fundamentals and applications. Menlo Park, Calif: Benjamin/Cummings, 1998.
2. Hanahan, D. and M. Meselson. Plasmid screening at high colony density. Gene. 1980 75. 1978
3. Lipps, G. Plasmids current research and future trends. Norfolk, U.K: Caister Academic P, 2008.
4. Paietta, J.V. and G.A. Marzluf Development of a colony hybridization technique for Neurospora. Proc. Natl. Acad. Sci. USA
5. White, Brian. "COLONY HYBRIDIZATION." MIT. 28 Apr. 2009 <http://filebox.vt.edu/users/chagedor/biol_4684/Methods/colhyb.html>.
Colony Hybridization
Introduction
Colony hybridization is an application of nucleic acid hybridization that is combined with conventional environmental microbiological sampling and viable planting procedures (1). This process utilizes a gene probes, which is radio actively marker, which attaches to complimentary base pairs from a single strand of bacteria DNA. This has proven to be a useful procedure for efficiently screening bacteria clones by RNA-DNA or DNA-DNA hybridization (4). This technique is accomplished by growing microbial communities from samples collecting at the research site on an agar plate, then transferring the colonies to filter paper to be lysed and denatured. After this step a labeled probe is added to bind to complementary strand of DNA and this product is assessed for any hybridization which is visualized as a black spot when looked at under x-ray. The original protocol was developed by Grunstein and Hogness (2).
Use of technology
This technique can be used to find similar microbial communities in a sample. If there is a known gene or sequence nucleic acids a researcher is looking for in a particular samples, whether it is taken from soil, water, or the air. This can also search for particular plasmids, which is extra chromosomal DNA (3). This technique allows a researcher to pick a single gene from the whole genome of a bacterium of interest, while at the same time saving the researcher from having to run DNA isolation assay or a PCR assay. The con’s of colony hybridization is that this assay is very time consuming, with all of the incubation this research takes days to complete. Another pitfall of this technique is that is only applies to microbes that can withstand the plating process, meaning 99% of the microbe species in the sample with not be incorporated into this experiment.
Materials and Protocol
1. Isolate and grow cultures in a suitable nutrient medium (5). Bacteria can be placed on selective agar for microbe colonies that are sensitive to plating; selective plates are used to facilitate growth of a particular microbe while inhibiting the growth of other microbes.
2. Transfer a sample of the colonies onto a solid matrix such as a nitrocellulose or nylon membrane (5). This procedure is done after the colonies have grown on the plate after incubation, which is usually done over night at 32oC however time and temperature can change depending upon the bacteria being assayed. The filter, which can be either Nylon or nitrocellulose filter, is placed on top of the bacterial colonies on the agar plate and incubated once the colonies have transferred to the filter for 24 hours at 30oC.3. The cells on the membrane are lysed and the DNA is then denatured (5). After incubation the filters were placed and incubated on a series of saturated papers that lyses the cell and denatures the DNA and helps it absorb into the filter paper. The first saturated paper contained 1M sorbitol, 40 mM EDTA with a PH of 8.0, and 50 mM DTT, this was incubated for 15 min at 30° C; the second saturated paper contans 1 M sorbitol, 40 mM EDTA with a PH of 8.0, and 10% glusulase, and incubated for 3-4 h, at 37° C; the third saturated paper contained 0.5 M NaOH, and 10x SSC, and was incubated for 8 min at 37° C; the fourth and fifth saturated papers contained 0.5 M Tris-HCl (pH 7.4), 10x SSC, and were both incubated for 4 min at 37° C (5).The filters were then transferred to a filtration block and, with suction applied, 200 ml of 3x SSC and 100 ml of chloroform were poured through the filter, and then the filters are baked in a oven at 80° C for 2 h(5).
4. A labeled gene probe is added to the matrix and hybridization takes place (5). The gene probe is made by isolating a piece of genetic information and the label is usually either 32P- or biotin. The hybridization occurs when the bases of the DNA match the sequence of the gene probe. When a gene probe finds its complementary DNA sequence they bind together and the specific genetic properties can be detected. This process begins with soaking the baked filter in 3x SSC for 15 minutes followed by 2 hours of incubation in the gene probe solution. Once the 2 hours incubation is completed the filter is then taken out and allowed to incubate at 65oC for 24 while the hybridizations are occurring (4).
5. The matrix is rinsed to remove the non-hybridized probe molecules (5). The wash reduces the level of background noise of the probe. The filter was washed with 3x SSC, then 2x SSC and lastly 1x SSC.
6. For radioactive probes, one uses autoradiography and the matrix is placed on x-ray film (5). This is when the colonies with the gene probe hybridization are visualized. If the colony has had a hybridizing event the bacteria will appear black under the X-ray autoradiograph.
7. Compare the x-ray film with the master plate to see which colonies had probe hybridization. These are the colonies that contained the specific sequence that actually hybridized with the probe (5). If everything is done properly it should be an easy task to compare the developed autoradiograph to the original plated bacterial colonies and to see which colony has had hybridization events.
Work Cited
1. Atlas, Ronald M. Microbial ecology fundamentals and applications. Menlo Park, Calif: Benjamin/Cummings, 1998.
2. Hanahan, D. and M. Meselson. Plasmid screening at high colony density. Gene. 1980 75. 1978
3. Lipps, G. Plasmids current research and future trends. Norfolk, U.K: Caister Academic P, 2008.
4. Paietta, J.V. and G.A. Marzluf Development of a colony hybridization technique for Neurospora. Proc. Natl. Acad. Sci. USA
5. White, Brian. "COLONY HYBRIDIZATION." MIT. 28 Apr. 2009 <http://filebox.vt.edu/users/chagedor/biol_4684/Methods/colhyb.html>.