Nucleic acid hybridization

This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.

1. Nucleic acid Hybridization Dr.N.C.J.Packia Lekshmi NICHE

2. Introduction DNA–DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two organisms. This has been used extensively in phylogeny and taxonomy. In order to compare DNA from different species, scientists use a technique called DNA hybridization.

3. 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 Watson‐Crick base pairs. Sequences that are closely related form base‐paired 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.

4. Principle DNA is double stranded, and individual bases bind based on Chargaff's rules of complementary bases: adenine pairs with thymine and cytosine pairs with guanine. When DNA is heated, the hydrogen bonds holding the base pairs together dissolve and the DNA separates into two single strands during a process known as DNA melting. The temperature at which the DNA strands separate is called the melting temperature. When DNA is cooled, complementary bases realign and bind through hydrogen bonding. During DNA-DNA hybridization, the DNA of each species is heated to the melting temperature to produce single strands. Then, a strand of DNA from each species is combined and allowed to anneal together (recombine). Then the sample is gradually heated. If the species DNA is very similar, they will form many hydrogen bonds through the base pairs and the melting temperature will need to be higher to separate the strands. If the species DNA is dissimilar, meaning the species are unrelated, the melting temperature will be lower because there will be fewer hydrogen bonds to break.

5. Procedure Single stranded target DNA is bound to a membrane support. DNA probe labeled with detector substance is added. DNA probe pairs with the complementary target DNA. Sequence of nucleotide in the target DNA can be identified.

6. A DNA probe or a gene probe is a synthetic, single-stranded DNA molecule that can recognize and specifically bind to a target DNA (by complementary base pairing) in a mixture of biomolecules . DNA probes are either long (> 100 nucleotides) or short (< 50 nucleotides), and may bind to the total or a small portion of the target DNA. There is a wide variation in the size of DNA probes used (may range from 10 bases to 10,000 bases). The most important requirement is their specific and stable binding with target DNAs.

7. DNA Probes synthesis A great majority of DNA probes are chemically synthesized in the laboratory. There are, however, many other ways of obtaining them-isolation of selected regions of genes, cloning of intact genes, producing from mRNAs. Synthesis of DNA probes from mRNA: The mRNA molecules specific to a particular DNA sequence (encoding a protein) are isolated. By using the enzyme reverse transcriptase, complementary DNA ( cDNA ) molecules are synthesized. This cDNA can be used as a probe to detect the target DNA. Isolation of selected regions of genes: The DNA from an organism (say a pathogen) can be cut by using restriction endonucleases . These DNA fragments are cloned in vectors and the DNA probes can be selected by screening.

8. Mechanism of Action of DNA Probes: The basic principle of DNA probes is based on the denaturation and renaturation (hybridization) of DNA. When a double-stranded DNA molecule is subjected to physical (temperature > 95°C or pH < 10.5) or chemical (addition of urea or formaldehyde) changes, the hydrogen bonds break and the complementary strands get separated. This process is called denaturation . Under suitable conditions (i.e., temperature, pH, salt concentration), the two separated single DNA strands can reassemble to form the original double-stranded DNA, and this phenomenon is referred to as renaturation or hybridization.

9. Detector system in DNA Hybridization Radioactive detector system Non-Radioactive detector system Radioactive Detection System: The DNA probe is usually tagged with a radioactive isotope (commonly phosphorus-32). The target DNA is purified and denatured, and mixed with DNA probe. The isotope labeled DNA molecules specifically hybridizes with the target DNA. The non-hybridized probe DNA is washed away. The presence of radioactivity in the hybridized DNA can be detected by autoradiography. This reveals the presence of any bound (hybridized) probe molecules and thus the complementary DNA sequences in the target DNA.

10. Non-radioactive Detection System: The disadvantage with the use of radioactive label is that the isotopes have short half-lives and involve risks in handling, besides requiring special laboratory equipment. So, non-radioactive detection systems (e.g., biotinylation ) have also been developed. Biotin-labeled ( biotinylated ) nucleotides are incorporated into the DNA probe. The detection system is based on the enzymatic conversion of a chromogenic ( colour producing) or chemiluminescent (light emitting) substrates. The procedure commonly adopted for chemiluminescent detection of target DNA. A biotin labeled DNA probe is hybridized to the target DNA. The egg white protein avidin or its bacterial analog streptavidin is added to bind to biotin. Now a biotin labeled enzyme, such as alkaline phosphatase is added which attaches to avidin or streptavidin . These proteins have four separate biotin-binding sites. Thus, a single molecule ( avidin or streptavidin ) can bind to biotin-labeled DNA probe as well as biotin-labeled enzyme. On the addition of a chemiluminescent substrate, the enzyme alkaline phosphatase acts and converts it to a light emitting product which can be measured.

11. The biotin-labeled DNA is quite stable at room temperature for about one year. The detection devices using chemiluminescence are preferred, since they are as sensitive as radioisotope detection, and more sensitive than the use of chromogenic detection systems.

12. PCR in the use of DNA probes: DNA probes can be successfully used for the identification of target DNAs from various samples — blood, urine, feces, tissues, throat washings without much purification. The detection of target sequence becomes quite difficult if the quantity of DNA is very low. In such a case, the polymerase chain reaction (PCR) is first employed to amplify the minute quantities of target DNA and identified by a DNA probe.

13. DNA Probes and Signal Amplification: Signal amplification is an alternative to PCR for the identification of minute quantities of DNA by using DNA probes. In case of PCR, target DNA is amplified, while in signal amplification it is the target DNA bound to DNA probe that is amplified. There are two general methods to achieve signal amplification. Separate the DNA target—DNA probe complex from the rest of the DNA molecules, and then amplify it. 2. Amplify the DNA probe (bound to target DNA) by using a second probe. The RNA complementary to the DNA probe can serve as the second probe. The RNA-DNA-DNA complex can be separated and amplified. The enzyme O-beta replicase which catalyses RNA replication is commonly used.

14. Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) is a laboratory method used to detect and locate a DNA sequence, often on a particular chromosome. In the 1960s, researchers Joseph Gall and Mary Lou Pardue found that molecular hybridization could be used to identify the position of DNA sequences in situ (i.e., in their natural positions within a chromosome). In 1969, the two scientists published a paper demonstrating that radioactive copies of a ribosomal DNA sequence could be used to detect complementary DNA sequences in the nucleus of a frog egg. Since those original observations, many refinements have increased the versatility and sensitivity of the procedure to the extent that in situ hybridization is now considered an essential tool in cytogenetics .

15. Principle of FISH Fluorescence in situ hybridization (FISH) is a technique that uses fluorescent probes which bind to special sites of the chromosome with a high degree of sequence complementarity to the probes. The fluorescent probes are nucleic acid labeled with fluorescent groups and can bind to specific DNA/RNA sequences. Thus, we can understand where and when a specific DNA sequences exist in cells by detecting the fluorescent group. It was developed in the early 1980s. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes and flow cytometry can be used to detect the binding quantitatively. This FISH protocol is for a Cy5 and FAM labeled probe used in flow cytometry detection and fluorescence microscopy detection.

16. Application An ideal technique for identification of: infectious diseases cancer genetic disorders mitochondrial disorders an aid to personalized and precision medicine based on the knowledge of pharmacogenomics .

17. Nucleic acid hybridization assay A hybridization assay comprises any form of quantifiable hybridization i.e. the quantitative annealing of two complementary strands of nucleic acids, known as nucleic acid hybridization. Hybridization assays involve labelled nucleic acid probes to identify related DNA or RNA molecules (i.e. with significantly high degree of sequence similarity) within a complex mixture of unlabelled nucleic acid molecules. Antisense therapy, siRNA , and other oligonucleotide and nucleic acid based biotherapeutics can be quantified with hybridization assays. Signalling of hybridization methods can be performed using oligonucleotide probes modified in-synthesis with haptens and small molecule ligands which act homologous to the capture and detection antibodies. As with traditional ELISA, conjugates to horse radish peroxidase (HRP) or alkaline phosphatase (AP) can be used as secondary antibodies.

18. Sandwich hybridization assay In the sandwich hybridization ELISA assay format, the antigen ligand and antibodies in ELISA are replaced with a nucleic acid analyte , complementary oligonucleotide capture and detection probes. Generally, in the case of nucleic acid hybridization, monovalent salt concentration and temperature are controlled for hybridization and wash stringency, contrary to a traditional ELISA, where the salt concentration will usually be fixed for the binding and wash steps (i.e. PBS or TBS). Thus, optimal salt concentration in hybridization assays varies dependent upon the length and base composition of the analyte , capture and detection probes.

19. Competitive hybridization assay The competitive hybridization assay is similar to a traditional competitive immunoassay. Like other hybridization assays, it relies on complementarity , where the capture probe competes between the analyte and the tracer–a labelled oligonucleotide analog to the analyte .

20. Hybridization-ligation assay In the hybridization-ligation assay a template probe replaces the capture probe in the sandwich assay for immobilization to the solid support. The template probe is fully complementary to the oligonucleotide analyte and is intended to serve as a substrate for T4 DNA ligase -mediated ligation. The template probe has in addition an additional stretch complementary to a ligation probe so that the ligation probe will ligate onto the 3'-end of the analyte . Albeit generic, the ligation probe is similar to a detection probe in that it is labelled with, for example, digoxigenin for downstream signalling . Stringent, low/no salt wash will remove un- ligated products. The ligation of the analyte to the ligation probe makes the method specific for the 3'-end of the analyte , ligation by T4 DNA ligase being much less efficient over a bulge loop, which would happen for a 3' metabolite N-1 version of the analyte , for example. The specificity of the hybridization-ligation assay for ligation at the 3'-end is particularly relevant because the predominant nucleases in blood are 3' to 5' exonucleases . One limitation of the method is that it requires a free 3'-end hydroxyl which may not be available when targeting moieties are attached to the 3'-end, for example. Further, more exotic nucleic acid chemistries with oligonucleotide drugs may impact upon the activity of the ligase , which needs to be determined on a case-by-case basis.

21. Dual ligation hybridization assay The dual ligation hybridization assay (DLA) extends the specificity of the hybridization-ligation assay to a specific method for the parent compound. Despite hybridization-ligation assay's robustness, sensitivity and added specificity for the 3'-end of the oligonculeotide analyte , the hybridization-ligation assay is not specific for the 5' end of the analyte . The DLA is intended to quantify the full-length, parent oligonucleotide compound only, with both intact 5' and 3' ends. DLA probes are ligated at the 5' and 3' ends of the analyte by the joint action of T4 DNA ligase and T4 polynucleotide kinase . The kinase phosphorylates the 5'-end of the analyte and the ligase will join the capture probe to the analyte to the detection probe. The capture and detection probes in the DLA can thus be termed ligation probes. As for the hybridization-ligation assay, the DLA is specific for the parent compound because the efficiency of ligation over a bulge loop is low, and thus the DLA detects the full-length analyte with both intact 5' and 3'-ends. The DLA can also be used for the determination of individual metabolites in biological matrices.

22. Nuclease hybridization assay The nuclease hybridization assay, also called S1 nuclease cutting assay, is a nuclease protection assay-based hybridization ELISA. The assay is using S1 nuclease, which degrades single-stranded DNA and RNA into oligo - or mononucleotides, leaving intact double-stranded DNA and RNA. In the nuclease hybridization assay, the oligonucleotide analyte is captured onto the solid support such as a 96-well plate via a fully complementary cutting probe. After enzymatic processing by S1 nuclease, the free cutting probe and the cutting probe hybridized to metabolites, i.e. shortmers of the analyte are degraded, allowing signal to be generated only from the full-length cutting probe- analyte duplex. The assay is well tolerant to diverse chemistries, as exemplified by the development of a nuclease assay for morpholino oligonucleotides . This assay set-up can lack robustness and is not suitable for validation following the FDA's guidelines for bioanalytical method validation