Title: Dot Blot DNA Hybridization. Introduction.

Transcription

1 Name: Date: Title: Dot Blot DNA Hybridization. Introduction. Complementary base pairing is the molecular basis of nucleic acid function. Replication, transcription, and translation all depend on hydrogen bonding between bases in nucleotides and nucleic acids. In replication and transcription nucleotides base pair with a DNA template strand to generate a new DNA strand or an RNA transcript. In translation nucleotides in the anticodons of trnas base pair with an mrna to bind the encoded amino acyl group to the ribosome for incorporation into a polypeptide chain. Complementary base pairing is also at the heart of many techniques used in modern genetics. It is the basis of techniques that replicate nucleic acid sequences for study, such as the polymerase chain reaction and in vitro transcription. It is also the basis of techniques that locate or quantify specific nucleic acid sequences, such as library screens, dot blots, DNA blots (Southern blots), RNA blots (Northern blots), and microarrays. The ability to locate and identify particular sequences using complementary base pairing depends on a phenomenon called hybridization. Any two nucleic acid molecules that have complementary base sequences can base pair, forming a double stranded hybrid molecule. Since this interaction depends on the base sequences of the nucleic acid molecules, it is highly specific. Hybridization can be used to locate and quantify a particular sequence. Essentially, nucleic acid molecules that include the sequence of interest are denatured and immobilized on a membrane. A nucleic acid probe that is complementary to the sequence of interest, and has been tagged with a readily detectable marker or label, is allowed to hybridize to the membrane-immobilized nucleic acid molecules. The probe will form a double stranded hybrid with the sequence of interest, binding the label to that part of the membrane that carries the sequence of interest. The label is then visualized, revealing the location of the sequence of interest on the membrane. Some Hybridization-Based Techniques. In library screens hundreds or thousands of bacteria or bacteriophage, each containing a different recombinant DNA molecule, are plated. DNA from the resulting colonies or plaques is denatured and immobilized on a membrane. To locate recombinant DNA molecules containing the sequence of interest, a probe is hybridized to the membrane. The label reveals the colonies or plaques that contain this sequence (see figure 1). For example, haemoglobin genes were first isolated by probing genomic DNA libraries with labelled mrna from erythroid cells. The genes have subsequently been used in many studies including investigations of the molecular basis of sickle cells anaemia and thalassemia. In dot blots DNAs or RNAs from different sources are denatured and spotted onto a membrane. To determine the amount of the sequence of interest in each of the spotted DNAs or RNAs, a probe is hybridized to the membrane. The more of the sequence of interest in the spotted DNA or RNA, the more probe will be bound to the spot. The amount of label reveals the amount of the sequence of interest in the spot (see figure 2). For example, excessive expression of certain oncogenes in tumour cells could be detected by spotting RNA from normal and tumour cells and probing with labelled oncogene sequences. Such studies have led to greater understanding of the molecular basis of cancer. Genetics Laboratory 20.1

2 Plate Membrane After Visualization Figure 1:Library screening. Bacteria or bacteriophage, each containing a different recombinant DNA molecule, are grown on a plate. The colony or plaque that contains the recombinant DNA molecule of interest (e.g., the haemoglobin genes) is indistinguishable from the other colonies or plaques on the plate. DNA from the resulting colonies or plaques is denatured and immobilized on a membrane. The arrow shows the position of a recombinant DNA molecule containing the haemoglobin gene. When the membrane is hybridized with a probe complementary to the haemoglobin gene it reveals the colony or plaque that contains this sequence. normal tumour RNA Spots Membrane After Visualization Figure 2: Dot blots. DNA or RNA from different sources (e.g., RNA from normal cells and RNA from tumour cells) are spotted on to a membrane. When the membrane is hybridized with a probe for the sequence of interest (e.g., an oncogene) the strength of signal produced reveals the amount of transcript produced from that oncogene. In DNA blots (Southern blots) DNA is cut with a restriction enzyme and the resulting fragments separated according to size by gel electrophoresis. The DNA fragments are denatured and transferred to a membrane. To locate the particular fragment or fragments that contain the sequence of interest, a probe is hybridized to the membrane. The position of the label reveals the size of the fragment or fragments that contain the sequence of interest (see figure 3). For example, the assembly of functional immunoglobulin genes from non-functional gene segments during development of an immune response was revealed by comparison of DNA blots (Southern blots) of DNA from immature and mature B lymphocytes probed with labelled immunoglobulin gene sequences. This has led to a much greater understanding of the ability of the immune system to respond to infection. In RNA blots (Northern blots) RNA molecules are separated according to size by gel electrophoresis. The RNA molecules are denatured and transferred to a membrane. To determine the amounts and sizes of RNAs that contain the sequence of interest, a probe is hybridized to the membrane. The position of the label reveals the size of the RNA or RNAs and the amount of label reveals the amount of each RNA (see figure 4). For example, the role of alternate splicing of primary transcripts in sex determination in Drosophila was first revealed by comparison of northern blots of mrnas from male and female flies probed with labelled DNA from genes which, when mutated, altered sexual phenotype. Genetics Laboratory 20.2

3 immature mature DNA Gel Membrane After Visualization Figure 3: DNA blots (Southern blots). DNA (e.g., genomic DNA from immature and mature B cells) is cut with a restriction enzyme and subjected to gel electrophoresis. The DNA fragment of interest (e.g., an immunoglobulin gene) is indistinguishable from the thousands of other DNA fragments. DNA from the gel is denatured and transferred to a membrane. The arrows show the position of the immunoglobulin gene fragment. When the membrane is hybridized with a probe complementary to the immunoglobulin gene it reveals that the size of the fragment that contains this gene changes as B cells develop. male female RNA Gel Membrane After Visualization Figure 4: RNA blots (Northern blots). RNA (e.g., from male and female Drosophila embryos) is subjected to gel electrophoresis. The RNA of interest (e.g., Sxl mrna) is indistinguishable from the thousands of other RNA molecules. RNA from the gel is transferred to a membrane. The arrows show the position of the Sxl mrna. When the membrane is hybridized with a probe complementary to the Sxl gene it reveals that the size of the mrna differs in the two sexes. Probes and Labels. In order to detect a probe bound by hybridization to a membrane, the probe must be tagged with a readily detectable marker or label. Labels can be incorporated into probes either by direct reaction of tag molecules with the probe nucleic acid or by in vitro duplication of the nucleic acid in the presence of tagged nucleotides. For example, DNA fragments can be incubated with primers, tagged nucleotides, and DNA polymerase. The DNA will be replicated and tagged nucleotides will be incorporated into the newly synthesized DNA. Similarly, mrna can be incubated with primers, tagged nucleotides, and reverse transcriptase. The mrna will be copied into DNA (complementary DNA or cdna) and tagged nucleotides will be incorporated into the newly synthesized cdna. Genetics Laboratory 20.3

4 Commonly used labels are radioactive isotopes of phosphorous or hydrogen, enzyme molecules, biotin, and fluorescent compounds. Each requires a different method for detection of membranebound probe. Probes labelled with radioactive isotopes are detected by placing photographic film over the membrane after hybridization. Particles produced by radioactive decay strike the photographic emulsion, producing a dark spot where the probe is bound to the membrane. This technique is called autoradiography. Radioactive probes allow for detection of minute amounts of complementary sequence; however, the procedures required for their safe use can be awkward. Consequently they are being superceded by recently developed non-radioactive probes that have similar sensitivity. Probes labelled with enzyme molecules are detected by soaking the filters in a solution that contains a chromogenic or luminescent substrate for the enzyme. Chromogenic substrates are converted into pigments, producing a coloured spot where the probe is bound to the membrane. Luminescent substrates emit light upon reaction, producing a glow that can be detected by placing photographic film over the membrane or by using an electronic detector. Probes labelled with biotin are detected by soaking the filters in a solution containing steptavidin-conjugated enzymes. Streptavidin binds to biotin, attaching the enzymes to the probe bound to the membrane. The enzymes can then be detected using chromogenic or luminescent substrates as described above. Probes labelled with fluorescent compounds are detected by illuminating the membrane with light at a wavelength that causes the fluorescent compound to fluoresce, producing a glow where the probe is bound to the membrane. This can be detected using an electronic detector. Some Factors Affecting Signal Strength. Assuming that there is an excess of probe in the hybridization, the strength of the signal produced will depend on the amount of complementary sequence present on the membrane. For example, if the amount of DNA spotted on a dot blot is doubled, the amount of a particular sequence in that DNA will be doubled, as will the amount of probe that will hybridize, Similarly, if identical amounts of genomic DNA are spotted on two dot blots, and one is hybridized with a probe for a sequence present just once in the genome, and the other with a probe for a repeated sequence, more of the repeated sequence probe will bind. However, there is an additional factor that must be considered. The relative stability of the double stranded hybrid molecule formed when a probe base pairs with its complementary sequence depends on the quality of the match between the two sequences 1. Sequences that match perfectly will form more stable hybrids than sequences that contain mismatches. This is reflected in the temperature at which the hybrid becomes unstable and the two strands separate. This temperature is known as the melting temperature of the hybrid molecule, and is abbreviated T m. 1 The stability of nucleic acid hybrids also depends on the percentage of guanine-cytosine base pairs (%G+C) in the complementary sequences and the salt concentration used for the hybridization. The higher the %G+C, the more hydrogen bonds between the two strands, and the more stable the hybrid. The higher the salt concentration, the more positively charged ions available to reduce repulsion between the negatively charged phosphate groups, and the more stable the hybrid. A more detailed discussion is given in the instructions for laboratory exercise 02. For many types of experiment involving nucleic acid hybrids the %G+C in the hybrid must be taken into account when deciding on the experimental conditions. Genetics Laboratory 20.4

5 As a rule of thumb, one percent mismatch between sequences reduces the melting temperature by one degree Celsius. The relationship between melting temperature and sequence mismatch can be used to control the quality of match that is required to generate a signal in a hybridization experiment. The higher the temperature at which the hybridization takes place, the better the match required to form a stable hybrid. If the temperature used is such that only a good match can form a stable hybrid molecule, the hybridization is said to have been done at high stringency. High stringency hybridization conditions will only detect sequences that match the probe almost perfectly. If the temperature used is such that a poor match can form a stable hybrid molecule, the hybridization is said to have been done at low stringency. Low stringency hybridization conditions will detect sequences that match the probe almost perfectly and sequences that are similar to the probe but have several mismatches. Hybridization and Evolutionary Relationships Hybridization experiments can be used to study evolutionary relationships. The more closely related two species are, the more similar their genomic DNA sequences will be. The more similar their genomic DNA sequences, the greater the number of hybrid DNA molecules that will be formed if the two genomic DNAs are denatured and mixed. Hence, if identical amounts of genomic DNA from several species are spotted on a dot blot and hybridized with a genomic DNA probe from another species, the dot containing the genomic DNA from the most closely related species will bind the greatest amount of probe and produce the strongest signal, and the dot containing the genomic DNA from the least closely related will bind the least amount of probe and produce the weakest signal. Methods. Day One In this laboratory session we will prepare dot blots by spotting genomic DNA from chicken, cow, salmon, and turkey onto nylon membranes. The dot blots will be hybridized with either a biotinylated chicken DNA probe or a biotinylated cow DNA probe. Each group will be responsible for preparation of two dot blots and hybridization of those dot blots with one of the two probe DNAs. IMPORTANT Wear gloves when handling DNA solutions and nylon membranes. Your skin has oils and proteins that will degrade DNA and interfere with DNA hybridization and probe visualization. Keep the DNA solutions on ice as much as possible. This will also prevent degradation of DNA. Note: All DNA solutions were denatured by heating at 100 C just before the laboratory session. (1) Take two strips of nylon membrane. Place them on a clean sheet of paper. Using a pencil, lightly draw a line across the membrane one centimeter from one end. Label one strip on the short side of the line with your initials and the letter A. Label the other strip on the short side of the line with your initials and the letter B. Genetics Laboratory 20.5

6 (2) Pour a small amount of distilled water into the plastic tray provided. Float the nylon membranes on the water and allow them to become wet. Submerge the membranes in the water. This procedure forces out air trapped in the membrane that might otherwise interfere with probe visualization. (3) Pour off the water in the tray and replace with approximately 5ml TBS buffer. Shake gently for a few seconds to allow the buffer to replace the water in the membrane. (4) Moisten a paper towel with distilled water and place on the bench. Remove the membranes from the TBS buffer and place on the damp paper towel. Blot the membranes with a paper towel to remove excess moisture. Place a ruler alongside the membranes. (5) Spot 5µl of each of the four genomic DNAs onto each membrane in the pattern shown in figure 5. The chicken DNA spot should be one centimeter from the line, the cow DNA spot should be two centimeters from the line, the salmon DNA should be three centimeters from the line, and the turkey DNA should be four centimeters from the line. initials pencil line chicken DNA cow DNA salmon DNA turkey DNA Figure 5: Labelling and arrangement of samples on nylon membrane. Wait 15 minutes to allow the DNA to be absorbed into the membrane. (6) Rinse the membranes briefly by dipping them in the tray containing TBS buffer. (7) Exchange one membrane with another group so that you either have two membranes labelled A or two membranes labelled B 2. Immediately place the two membranes into a hybridization bag, making sure that one end of each membrane is at the bottom of the bag. (8) Add 3ml of hybridization buffer to the bag. Close the seal, fold over the top 2.5cm of the bag and secure with paper clips as shown in figure 6. 2 One membrane from the class will be stained with methylene blue for 10 minutes to determine the relative amounts of DNA in the four spots. You should examine the stained membrane and record the relative intensities of the four DNA spots. Genetics Laboratory 20.6

7 fold paper clip paper clip closure Figure 6: Method used for sealing hybridization bag. Close the bag, then fold and secure with paper clips at the points indicated. (9) Empty the tray that you used to wet the membranes and dry it with paper towels. Lay the hybridization bag flat in the tray. Place the lid on the tray. (10) Float the tray in the 65 C water bath. Incubate for 15 minutes. This step is called pre-hybridization. Reagents in the buffer bind to the membrane, reducing non-specific binding of the probe. (11) Remove the tray from the water bath. Bend the paper clips to remove them from the bag. (Do not slide the paper clips off as this may puncture the bag.) Discard the hybridization buffer into the beaker provided. (12) Add 3.5ml of fresh hybridization buffer to a test tube. If your membranes are labelled A, add 50µl of biotinylated chicken DNA probe to the tube. If your membranes are labelled B, add 50µl of biotinylated cow DNA probe to the tube. (13) Gently pour the hybridization buffer containing the biotinylated probe DNA into the bag containing the membranes. Remove as much air as possible from the bag, then seal it as described above in step 8 (figure 6). (14) Return the sealed bag to the tray, place the lid on the tray, and return the tray to the 65 C water bath. The membranes will be allowed to hybridize with the probes for 18 to 24 hours. The bags will then be removed and the membranes washed to remove any probe that has not hybridized. The washed membranes will be stored at 4 C until the next laboratory session. Genetics Laboratory 20.7

8 Day Two In this laboratory session we will visualize the hybridized probe DNA using avidin-peroxidase and a chromogenic substrate. Each group will visualize the two membranes they prepared in the previous session. IMPORTANT Wear gloves when handling nylon membranes. Your skin has oils and proteins that will interfere with probe visualization. Keep the avidin-peroxidase and chromogenic substrate solutions on ice as much as possible. (1) Get the two membranes that you prepared in the previous session. Place them into a hybridization bag, making sure that one end of each membrane is at the bottom of the bag. (2) Add 3ml of TBS-gelatin solution and 25µl of avidin-peroxidase to a test tube. Gently pour this mixture into the bag containing the membranes. Remove as much air as possible from the bag, then seal it as described previously (day 1, step 8 (figure 6)). (3) Lay the hybridization bag flat in a plastic tray. Place the lid on the tray. Float the tray in the 37 C water bath. Incubate for 40 minutes. During this step the avidin-peroxidase will bind to the biotin attached to the probe DNA. (4) Remove the membranes from the bag and place them in the tray. (5) Add 10ml TBS buffer to the tray and incubate at room temperature for five minutes. This step and the two that follow remove any unbound avidin-peroxidase and prepare the membranes for the final colour development step. (6) Discard the TBS buffer. Add 15ml TBS-NP40 buffer to the tray and incubate at room temperature for five minutes. (7) Discard the TBS-NP40 buffer. Add 15ml CD buffer to the tray and incubate at room temperature for five minutes. (8) Discard the CD buffer. Add 10ml chromogenic substrate solution to the tray. Gently rock the tray at room temperature for about 15 minutes. During this step the peroxidase bound to the probe DNA will convert the chromogenic substrate into a purple pigment. (9) Discard the chromogenic substrate solution. Rinse the membranes in distilled water and record the relative intensities of the purple spots on the membranes. (10) Blot the membranes with a paper towel to remove excess moisture. Place the membranes in a folded paper towel and store them in a folder or notebook. Genetics Laboratory 20.8

9 Report. Your report should be typed or neatly written. Marks may be deducted for illegibility and/or poor grammar and/or poor spelling. Your report should be stapled once in the top left corner. Binders or paper clips are not acceptable. Your report should be in the following format: Name and date. Title of experiment. Introduction to experiment. Description of methods and materials used. The above information is in this manual. You can attach the manual pages to your report in place of this information. Results obtained. Report the intensity of the colour observed for each spot on your membranes that were probed with chicken DNA and cow DNA, and for each spot on the membrane that was stained for DNA. Note which probe you used for hybridization, and give the names of the students who probed your second membrane with the alternate probe. Analysis of results obtained and conclusions. Using only the data from hybridization with the chicken DNA probe, rank the four species (chicken, cow, salmon, turkey) in terms of their sequence similarity to chicken. Using only the data from hybridization with the cow DNA probe, rank the four species (chicken, cow, salmon, turkey) in terms of their sequence similarity to cow. Discussion. Were the amounts of chicken, cow, salmon, and turkey DNA spotted onto the membrane equal? Does this change your ranking of the four species in terms of their sequence similarity to chicken and to cow? If so, how are the rankings changed? Are these rankings in agreement with your expectations? Comment on any discrepancies. Construct an evolutionary tree showing the history of the four species based on your data. Is this tree in agreement with your expectations? Comment on any discrepancies. You wish to detect a gene using hybridization. The only probe DNA available comes from a distantly related species. Should you carry out the hybridization at a high or low temperature? Explain your answer. You wish to detect a gene using hybridization. The gene is one of a family of genes of related sequence. The probe DNA is most similar in sequence to the gene in which you are interested, but it does match the other genes in the family to a significant extent. Should you carry out the hybridization at a high or low temperature? Explain your answer. Genetics Laboratory 20.9

10 The report is due one week from today. Late reports will be penalized. You should each write your own report, although you may discuss your results with each other. If you require any help with your report, please come and see me. There is no penalty attached to this help. Genetics Laboratory 20.10