Nucleic acid hybridization.pptx molecular biology

Nucleic acid hybridization

1. Nucleic Acid Hybridization By Asmaa Kenawy , p h d Human Genetics Department

2. - When two single-stranded nucleic acid molecules of complementary base sequence form a double-stranded hybrid , the process is known as Nucleic Acid Hybridization - Can occur between: DNA-DNA RNA-RNA DNA-RNA Nucleic Acid Hybridization

3. Nucleic Acid Hybridization Applications PCR: hybridization between primers and the template DNA In Situ Hybridization DNA Microarrays MLPA/ MS-MLPA

4. Probe Probe: A labelled molecule that binds specifically to the molecule of interest, used for detection of specific DNA in a mixture of DNA fragments or total cell DNA. Labelling of this complementary DNA strand by: Radioactive Atom Fluorescent Tag Enzyme

5. Probe When this probe is added to the sample containing the target sequence it will bind to it because of the complementary base pairing Once bound, we can detect the bounded probe by the visualization or detecting the label If radiolabeled, it is detected by autoradiography If labeled with fluorescent tag, it is detected by fluorescence If labeled with enzyme, it is detected by colour (Chromogenic) or light (chemiluminescent) forming reactions catalyzed by the enzyme

6. Blotting Techniques

7. What is Blotting technique?

8. Southern Blotting A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization . The method is named after its inventor, the British biologist Edwin Mellor Southern (1975). Sir Edwin Mellor Southern

9. Southern Blotting The key to this method is hybridization . Hybridization: It is the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA.

10. Southern Blotting steps Step 1 DNA digestion. Step 2 Gel electrophoresis. Step 3 Blotting. Step 4 Probe labeling. Step 5 Hybridization & washing. Step 6 Detection.

11. Southern Blotting Procedure In the first step, sample DNA is broken down or digested in to smaller pieces using a restriction enzyme. After digestion, the DNA fragments are separated using Agarose gel electrophoresis. Electrophoresis shows several bands that look like a smear due to the presence of several small restriction fragments in the gel. Alkaline buffer ( NaOH ) is then used to denature the DNA into single strands.

12. Southern Blotting Procedure After electrophoresis, DNA is transferred to a positively charged nylon or nitrocellulose membrane. Traditional transfer of DNA is done overnight using an upward-transfer method/ capillary transfer During hybridization, the labeled probe is incubated with the DNA fragments that are immobilized on the blot under conditions that promote hybridization of complementary sequences

13. Southern Blotting Procedure After hybridization, the unhybridized probe is removed by washing in several changes of buffer. The probe is usually labeled with a chemical or radioactive tag so as to enable tracking of the probe on the gel. Chemical substrates and X-ray films are used to locate the probe in case the tag is an enzyme. Radioactive tags can directly show up on x-ray films.

14. Southern Blotting Applications identification of a single gene in a pool of DNA fragments gene mapping analysis of genetic patterns of DNA detection of specific DNA sequences in a genome study of gene deletions, duplications, and mutations that cause various diseases detection of genetic diseases and cancers such as monoclonal leukemia and sickle cell mutations detect the presence of a gene family in a genome DNA fingerprinting and forensic tests such as paternity testing and sex determination

15. Southern Blotting disadvantages requires a large amount of target DNA Complex Time consuming Radioactive probes

16. Southern Blotting https://www.youtube.com/watch?v=-SaKbLTQ4WI

17. Northern Blotting The northern blot technique is used to study gene expression by detection of RNA (or isolated mRNA) in a sample. This technique was developed in 1977 by James Alwine , David Kemp and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot.

18. Northern Blotting steps RNA is isolated from several biological samples (e.g. various tissues , various developmental stages of same tissue etc .)* RNA is more susceptible to degradation than DNA . Sample’s are loaded on gel and the RNA samples are separated according to their size on an agarose gel The resulting gel following after the electrophoresis run.

19. Northern Blotting steps In the Southern blotting, DNA fragments are denatured with alkaline solution. In the Northern blotting, RNA fragments are treated with formaldehyde to unfragment the branched RNA molecule to simple linear one and to prevent it form coiling again.

20. Northern Blotting steps The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement. The membrane is placed in a dish containing hybridization buffer with a labeled probe. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. The membrane is washed to remove unbound probe .

21. Northern Blotting steps The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film. The expression patterns of the sequence of interest in the different samples can be compared.

22. Northern Blotting APPLICATIONS A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts). Detection of mRNA transcript size . Study RNA degradation . Study RNA splicing . Study RNA half-life. Often used to confirm and check transgenic /knockout mice (animals) .

23. Northern Blotting Disadvantages The standard northern blot method is relatively less sensitive than nuclease protection assays and ( qPCR). Detection with multiple probes is a problem. If RNA samples are even slightly degraded by RNAses , the quality of the data and quantitation of expression is quite negatively affected. Time consuming procedure . Use of radioactive probes .

24. Northern Blotting https:// www.youtube.com/watch?v=G2Z1wuEOZUU

25. Western Blotting (Immunoblotting) Western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin , et al. in 1979. A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies The specificity of the antibody-antigen interaction enables a target protein to be identified in a complex protein mixture. Western blotting can produce qualitative and semi-quantitative data about the protein of interest, used to isolate , identify and quantify proteins in biological sample

26. Western Blotting (Immunoblotting) PROCEDURE Tissue preparation Gel electrophoresis Transfer Blocking Probing with antibodies Detection

27. Western Blotting

28. Western Blotting (Immunoblotting) Gel electrophoresis The first step in a western blotting procedure is to separate the macromolecules in a sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) , It separates proteins according to mass due to the negative charge imparted on proteins bound to the ionic SDS detergent .

29. Western Blotting (Immunoblotting) Transfer Subsequently , the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The transfer method that is most commonly used for proteins is electroelution or electrophoretic transfer because of its speed and transfer efficiency. This method uses the electrophoretic mobility of proteins to transfer them from the gel to the membrane.

30. Western Blotting (Immunoblotting) Transfer Electrophoretic transfer of proteins involves placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose or other suitable, protein-binding support and "sandwiching" this between two electrodes submerged in a conducting solution.

31. Western Blotting (Immunoblotting) Transfer The process involves the use of porous pads and filter paper to facilitate the transfer. When an electric field is applied, the proteins move out of the polyacrylamide gel and onto the surface of the membrane, where the proteins become tightly attached. The result is a membrane with a copy of the protein pattern that was originally in the polyacrylamide gel.

32. Western Blotting (Immunoblotting) Blocking After the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane.

33. Western Blotting (Immunoblotting) Probing with antibodies Most commonly, the transferred protein is then probed with a combination of antibodies: one antibody specific to the protein of interest ( primary antibody ) and another antibody specific to the host species of the primary antibody ( secondary antibody ). Often the secondary antibody is complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal.

34. Western Blotting (Immunoblotting) Detection Procedures vary widely for the detection step of a western blot experiment. One common variation involves direct versus indirect detection. With the direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot. This detection method is not widely used.

35. Western Blotting (Immunoblotting) In the indirect detection method, an unlabeled primary antibody is first used to bind to the antigen. Subsequently, the primary antibody is detected using an enzyme- or fluororophore -conjugated secondary antibody. Labels (or conjugated molecules) may include fluorescent probes, and enzyme conjugates such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) .

36. Western Blotting (Immunoblotting) 1. Chromogenic substrates produce a precipitate on the membrane resulting in colorimetric changes visible to the eye. 2. The most sensitive detection methods use a chemiluminescent substrate that produces light as a byproduct of the reaction with the enzyme conjugated to the antibody. The light output can be captured using film . However, digital imaging instruments based on charge-coupled device (CCD) cameras are becoming popular alternatives to film for capturing chemiluminescent signal.

37. Western Blotting (Immunoblotting) 3. Alternatively , fluorescently tagged antibodies can be used, which require detection using an instrument capable of capturing the fluorescent signal. Fluorescent blotting is a newer technique and is growing in popularity as it affords the potential to multiplex (detect multiple proteins on a single blot). Whatever system is used, the intensity of the signal should correlate with the abundance of the antigen on the membrane.

38. Western Blotting (Immunoblotting)

39. Western Blotting (Immunoblotting)

40. Western Blotting (Immunoblotting) https:// www.youtube.com/watch?v=Ll_7z4YS2Ak&t=307s https:// www.thermofisher.com/eg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html

41. SDS – PAGE https:// www.youtube.com/watch?v=DDOGADWIHBw NATIVE PAGE https:// www.youtube.com/watch?v=5obiHqeYEc0 Primary and Secondary Antibodies (FL- Immuno /69) https:// www.youtube.com/watch?v=qQ0BaZ83ZtY

42. Microarray

43. DNA microarray A DNA microarray also commonly known as DNA chip or biochip is a collection of microscopic DNA spots attached to solid support surface. Each DNA spot contain specific DNA sequence known as probe or oligos . Each known gene or probe occupied a specific site on the chip and varying level of fluorescent activity show varying level of gene activity of introduced genetic material.

44. PRINCIPLE The core principle of microarray is HYBRIDIZATION. Samples are labelled using fluorescent dyes. Complementary nucleic acid get bind via hydrogen bonds. Washing of non specific bonding DNA . Scanning and data analysis

45. Types of Microarrays 1. Microarray Expression Analysis (cDNA microarrays): It is a laboratory tool used to detect the expression of thousands of genes at the same time. It enables large-scale analysis of mRNA abundance as an indicator of gene expression.

46. Types of Microarrays 1. Microarray Expression Analysis (cDNA microarrays): In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease

47. https:// www.youtube.com/watch?v=6ZzFihESjp0 https:// www.youtube.com/watch?v=pWk_zBpKt_w

48. Types of Microarrays 2. Single nucleotide polymorphism (SNP) microarrays and mutation arrays : To detect polymorphisms or mutations within a population using SNP arrays or arrays designed to detect known mutations. A SNP, a variation at a single site in DNA, is the most frequent type of variation in the genome, at frequencies of 1% or higher across different populations worldwide

49. Types of Microarrays 2. Single nucleotide polymorphism (SNP) microarrays and mutation arrays : The three mandatory components of the SNP arrays are : An array containing immobilized allele-specific oligonucleotide (ASO) probes: Two probes must be used for each SNP position to detect both alleles. Fragmented nucleic acid sequences of target, labelled with fluorescent dyes. A detection system that records and interprets the hybridization signal.

50. Types of Microarrays 2. Single nucleotide polymorphism (SNP) microarrays and mutation arrays : An SNP array is a useful tool for studying slight variations between whole genomes. The most important clinical applications of SNP arrays are for determining disease susceptibility and for measuring the efficacy of drug therapies designed specifically for individuals . In research, SNP arrays are most frequently used for genome-wide association studies SNPs can also be used to study genetic abnormalities in cancer. For example, SNP arrays can be used to study loss of heterozygosity (LOH).

51. Types of Microarrays 3. Array comparative genomic hybridization (array CGH, aCGH ): it is a molecular cytogenetic technique for the detection of chromosomal copy number variations (CNVs ) on a genome wide and high-resolution scale. It is used to identify genomic gains and losses, or a change in the number of copies of a particular gene involved in a disease

52. Types of Microarrays 3. Array comparative genomic hybridization (array CGH, aCGH ): Array CGH compares the patient's genome against a reference genome and identifies differences between the two genomes, and hence locates regions of genomic imbalances in the patient https :// www.youtube.com/watch?v=ZKVeMLsS7KQ&feature=youtu.be&fbclid=IwAR3pNUIWl9osZ9OYArCVpAlbv8vm-FCJD5jz_1CxDKLszqbJArzHVWsCoig

53. Types of Microarrays 3. Array comparative genomic hybridization (array CGH, aCGH ): Resolution indicates the number of probes on a microarray platform. The number of probes determines the resolution with which you can view the genome. That is, as the number of probes increases the coverage for the genome increases and the space between the probes decreases.

54. Types of Microarrays 3. Array comparative genomic hybridization (array CGH, aCGH ): When the space between the probes decreases, it will enable you to detect micro deletions and amplifications (focal aberrations). The 60K resolution arrays have 60,000 probes with a space of 40kb between the probes. While for an example a 40K array will have 40,000 probes and the space of 70kb between the probes.

55. Applications of Microarrays Gene Discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions . Disease Diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study and classification of cancer.

56. Applications of Microarrays Drug Discovery: Microarray technology has extensive application in Pharmacogenomics. Toxicological Research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.

57. Microarrays

58. Multiplex ligation-dependent probe amplification (MLPA) Multiplex Ligation-dependent Probe Amplification (MLPA) is a semi-quantitative technique that is used to determine the relative copy number of up to 60 DNA sequences in a single multiplex PCR-based reaction.

59. MLPA ASSAY PRINCIPLE The principle of MLPA is based on the amplification of up to 60 probes, each of which detect a specific DNA sequence of approximately 60 nt in length. The MLPA reaction results in a set of unique PCR amplicons approximately between 64-500 nt in length, which are separated by capillary electrophoresis.

60. MLPA steps Denaturation Hybridization Ligation Amplification (by PCR) Fragment Separation and Data Analysis

61. 1-Denaturation and 2 – Hybridization Denaturation involves separation of the annealed DNA strands, so that double-stranded DNA becomes single-stranded . Hybridization involves hybridizing the DNA sample to specific probes. Because it is a multiplex technique, you can analyze each sample by up to 60 probes simultaneously, thus targeting different sites

62. 3-Ligation The ligation step will bind the two probes together. In this step, a specific enzyme called DNA ligase is used. It binds the probes that are already hybridized on adjacent sites of the DNA strand at the target site The enzyme ligase is extremely specific: if there are any mismatches between the probe and the target site, the ligase will not be able to bind the probes and no amplification would occur.

63. 4-Amplification The next step is amplification, which is essentially a polymerase-chain reaction (PCR ). For the PCR step, a polymerase, dNTPs , and a forward and reverse primer are added. Since all of the probes have the same PCR-primer sequence, it will only be necessary to add one pair of universal primers to study all of the targets. The forward primer is fluorescently labelled, allowing visualization and quantitation during analysis.

64. 5-Fragment Separation and Data Analysis After amplification, the fragments are separated by capillary electrophoresis . Capillary electrophoresis separates fragments based on their length, and shows different length fragments as peak patterns, called an electropherogram . Each amplicon has a different known size, due to the stuffer sequence on each specific probe, and therefore each amplicon can be quantified during data analysis . The data obtained by capillary electrophoresis will be the input for the analysis. MRC- Holland provides a free software for data analysis – Coffalyser

65. 5-Fragment Separation and Data Analysis By comparing each sample to a set of reference samples, we can obtain a probe ratio. This probe ratio will inform us of how many copy numbers a gene has. Since most human genes are diploid, if the sample presents two copies, the ratio will be 1.0 ; i.e. the sample probes have obtained the same number of genes as the reference sample . However, if the ratio is 0.5 there was only one copy of the gene in the individual, which probably means a heterozygous deletion of the target gene. If, on the other hand, the ratio is 1.5 , there is, probably, a heterozygous duplication of a gene .

66. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) is a semi-quantitative, non-automated technique that is used to determine the relative copy number and methylation status for multiple targets simultaneously , up to 60 DNA sequences in a single multiplex PCR-based reaction . Copy number variations (CNVs) and DNA methylation aberrations in human DNA play a role in a large number of disorders .

67. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) To determine both copy number and methylation status of the target DNA, MS-MLPA probemixes contain several methylation-specific probes. These are designed to target DNA sequences which contain a restriction site for the methylation-sensitive restriction enzyme HhaI .

68. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) After probe hybridization, the MS-MLPA reaction is split into two parts . One part of the MS-MLPA reaction is processed as a normal MLPA reaction, providing information on copy number status of the target DNA . The other part is treated with the HhaI enzyme, which provides information on methylation status of the target DNA

69. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) When hybridising to an unmethylated DNA target, the methylation-specific probes will be ligated and simultaneously digested by HhaI . A digested MS-MLPA probe will not generate a peak signal because it cannot be amplified. In contrast, when the target sequence of the MS-MLPA probe is methylated, the methyl group will prevent HhaI -digestion. An undigested, ligated probe can be amplified during PCR , resulting in a normal peak signal.

70. Advantages of MLPA MLPA is a highly sensitive and high throughput technique . MLPA reaction is fast, inexpensive and very simple to perform It can discern between point mutations, as well as duplication/deletion of genes. Results are available within 24 hours and because it is a multiplex reaction, it allows for a quick and efficient gathering of information. Small alterations to the MLPA protocol can allow for a variety of applications. For example, by adding an extra digestion step, MLPA can also be used to detect methylation patterns in DNA ( Methylation specific-MLPA (MS-MLPA)).

71. Advantages of MLPA MLPA has a variety of applications including detection of mutations and single nucleotide polymorphisms , analysis of DNA methylation , relative mRNA quantification , chromosomal characterisation of cell lines and tissue samples , detection of gene copy number , detection of duplications and deletions in human cancer predisposition genes such as BRCA1, BRCA2 and aneuploidy determination

72. Limitations of MLPA For most MLPA applications, most of small (point) mutations will not be detected by MLPA probemixes . MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect copy number neutral inversions or translocations. Even when MLPA did not detect any aberration, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected . MLPA is extremely sensitive to impurities

73. Limitations of MLPA Sequence changes (e.g. SNPs, point mutations, small indels ) in the target sequence detected by a probe can cause false positive results. Mutations/SNPs (even when >20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA.