Hybridization is a technique in which molecules of single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) are bound to complementary sequences of either single-stranded DNA or RNA. Complementary base pairs are adenine (A) with thymine (T) or uracil (U) and vice versa, and guanine (G) with cytosine (C) and vice versa. Although the DNA double helix is relatively stable at body temperatures, high temperatures can split, or "melt," the double helix into single, complementary strands. After disrupting the double helix in this way, lowering the temperature then causes the single-stranded DNA to base-pair, or anneal, to other single strands that have complementary sequences. Single-stranded DNA can hydridize to either single-stranded DNA or single-stranded RNA. Two complementary single-stranded DNA molecules can reform the double helix after annealing. In DNA-RNA hybridization, the RNA base uracil pairs with adenine in DNA. Single-stranded RNA that is complementary to a messenger RNA (mRNA) sequence is called "antisense" RNA. Antisense RNA and mRNA form a double helix that is slightly different from a DNA double helix. Researchers use hybridization for many purposes. Overall genetic relatedness of two species can be determined by hybridizing their DNA. Due to sequence similarity between closely related organisms, higher temperatures are required to melt such DNA hybrids when compared to more distantly related organisms. In forensic DNA testing, a variety of different methods use hybridization to pinpoint the origin of a DNA sample, including the polymerase chain reaction (PCR). PCR produces many copies of a particular nucleic acid sequence and is also used to clone genes. In another technique, short DNA sequences are hybridized to cellular mRNAs to identify expressed genes. Pharmaceutical drug companies are exploring the use of antisense RNA to bind to undesired mRNA, preventing the ribosome from translating the mRNA into protein .

Read more: http://www.biologyreference.com/Ho-La/Hybridization.html#ixzz3qAMM4uxh

DNA Hybridization Nucleic acid hybridization allows scientists to compare and analyze DNA and RNA molecules of identical or related sequences. In a hybridization experiment, the experimenter allows DNA or RNA strands to form WatsonCrick base pairs. Sequences that are closely related form basepaired double helices readily; they are said to be complementary. The amount of sequence complementarity is a measure of how closely the information of two nucleic acids relate. The complementary strands can be both DNAs, both RNAs, or one of each.Heating the DNA solution above a characteristic temperature can separate the two strands of a double helix. That temperature is called the melting temperature, abbreviated T m . Above the T m, a DNA is mostly or all singlestranded; below the T m, it is mostly doublestranded. For a natural DNA, the T m depends primarily on its G+C content. Because a GC base pair has three hydrogen bonds and an AT pair only has two, nucleic acid double helices with a high G+C content have a higher T m than do those with a greater proportion of A+T. The T m is not an exact property: It depends on the solvent conditions. For example, a high concentration of salt (such as NaCl) raises the T m of a DNA duplex, because the positive Na+ ions shield the negative charges on the phosphodiester backbone from repelling each other. Likewise, certain organic solvents can cause the negative charges on the phosphates to repel more strongly; these solvents lower the T m of a DNA double helix. What happens if two nucleic acids are partly complementary and partly different? In this case, some stretches of the two strands may form base pairs while others don't. The two molecules can be manipulated so that they form a hybrid or separate. The conditions favoring the formation of duplex nucleic acid are low temperature (below the T m), high salt, and the absence of organic solvents. The latter two conditions raise the T m of the hybrid duplex so that the DNA would remain more doublestranded. On the other hand, higher temperatures (closer to the T m of the hybrid) lower salt, and the presence of organic solvents would tend to push the two strands of the DNA apart. The term stringency sums up these variables: The more stringent the conditions, the more likely partially complementary sequences are to be forced apart. Conversely, less stringent hybridization conditions mean that the two strands need not be so complementary to form a stable helix. See Figure 1.

Figure 1

Hybridization can be used to classify the DNAs of various organisms. For example, human DNA is 98 percent identical to that of chimpanzees, and these two DNAs form a duplex under stringent conditions. Related sequences of humans and birds can also form hybrids, but only at a much lower stringencyhttp://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/molecular-cloning-of-dna/dna-hybridization

Sandwich DNA Hybridization Assays (GENE-TRAK pathogen test kits in dipstick and microwell formats, and GeneQuence test systems in microwell format, excluding GeneQuence E. coliO157:H7)Each test kit contains capture and detector DNA probes specific to ribosomal RNA (rRNA) of the target organism and a coated solid phase (dipstick or microwell).First, a portion of the enrichment culture is placed into a test tube. A lysis reagent is added, which disrupts the cell and releases the nucleic acid target molecules. A portion of the lysed sample is then transferred to a microwell and the probe reagents are added.(In the dipstick format assay, the probe reagents are added to the tube with the lysed sample, followed by introduction of the coated dipstick to the tube.)The probe reagents consist of: 1) an oligonucleotide capture probe specific to rRNA sequences of the target organism and labeled at the 3 end with polydeoxyadenylic acid (poly dA); and 2) an oligonucleotide detector probe also specific to rRNA sequences of the target organism and labeled at the 5 end with the enzyme horseradish peroxidase (HRP). The hybridization reaction is then allowed to proceed for one hour. If target rRNA is present in the sample, both probes will hybridize to their complementary sequences on the target molecule. The resulting complex is captured onto the solid phase coated with polydeoxythymidylic acid (poly dT), which is complementary to the poly dA portion of the capture probe. Unbound probe is then washed away, and a substrate of HRP is added. Following a short incubation, blue color indicates the presence of hybridized detector probe in the complex and thus the presence of rRNA from the target organism. Results are determined spec-trophotometrically at 450 nm. An absorbance value in excess of a predetermined threshold indicates a positive test result.

http://www.neogen.com/FoodSafety/pdf/DNA_H_Diagram.pdf

Southern BlottingSouthern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.This diagram shows the basic steps involved in a Southern blot.

Let's look at this technique in greater detail. 1. Digest the DNA with an appropriate restriction enzyme.2. Run the digest on an agarose gel.3. Denature the DNA (usually while it is still on the gel).For example, soak it in about 0.5M NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.

A depurination step is optional. Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. Be aware, however, that the procedure may also be hampered by fragments that are too small.Be sure to neutralize the acid after this step, or the base after the prior step if you don't depurinate.4. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. Nitrocellulose typically has a binding capacity of about 100g/cm, while nylon has a binding capacity of about 500 g/cm. Many scientists feel nylon is better since it binds more and is less fragile. Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.

You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, excepts more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)

After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)5. Probe the membrane with labeled ssDNA. This is also known as hybridization.Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.

A prehybridization step is required before hybridization to block non-specific sites, since you don't want your single-stranded probe binding just anywhere on the membrane.

To hybridize, use the same buffer as for prehybridization, but add your specific probe.6. Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent visualization uses luminesence.32P labeled ATPTreat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in DNA. Add 32P, dATP, and other dNTPs to DNA polymerase I, which has 5' to 3' polymerase activity and 5' to 3' exonuclease activity.Nick translation occurs and as the nick is translated down the DNA strand, the polymerase activity continues to nick while the exonuclease activity continues to fill in the nick. As this happens, 32P becomes incorporated into, and thus labels, the DNA. Heat the DNA to make it single stranded, then immediately place it on ice to keep the two strands from reannealing to each other. (If the DNA is on ice, the DNA passes through the annealing temperature too quickly for the DNA to rehybridize into double-stranded DNA.)Prehybridization To prehybridize, add non-specific ssDNA. Somicated salmon sperm DNA is commonly used. Add 20X SSC, Denhardt's solution (ficol and PVP, which are large molecules to take up space and generate more contact; and BSA, bovine serum albumen, a non-specific protein), SDS (sodium dodecyl sulfate), and formamide.Altering the concentrations of formamide, SSC, and SDS affects "stringency," or specificity. If you have a higher stringency you should also have a higher degree of similarity between the probe and the target sequencehttps://askabiologist.asu.edu/southern-blotting