7. DNA Reassociation.pptx Dr Thirunahari Ugandhar

Dr Thirunahari Ugandhar

1. DNA Reassociation by Dr. Thirunahari Ugandhar Associate Prof of Botany Department of Botany Kakatiya Govt College (A) Hanamkonda

2. Steps in DNA Reassociation 1.Denaturation: Definition: The initial step where double-stranded DNA is melted into single strands. Method: Achieved by heating DNA to high temperatures or using denaturing agents like urea or formamide. Result: Hydrogen bonds between complementary base pairs (A-T and G-C) are broken, yielding ssDNA. 2.Annealing: Definition: Single-stranded DNA is gradually cooled under controlled conditions. Mechanism: Complementary sequences in the ssDNA align and form hydrogen bonds, re-establishing base pairs (A pairs with T, and G pairs with C). 3.Reassociation: Process: Complementary ssDNA molecules come together to form dsDNA duplexes. Factors Influencing Reassociation: DNA concentration: Higher concentrations promote reassociation. Temperature: Gradual cooling facilitates annealing. Sequence length and complexity: Longer and simpler sequences reassociate more readily

3. Applications of DNA Reassociation 1. Genome Complexity: Estimation of genome size and identification of repetitive elements. Analysis of DNA sequence organization and evolutionary patterns. 2. Evolutionary Studies : Comparison of genome similarity among species to infer evolutionary relationships. 3. Molecular Techniques : Basis for DNA hybridization assays, Southern blotting, and DNA microarray analysis.

4. Significance DNA reassociation is a foundational tool in molecular biology and genetics research. It helps in: Understanding genome organization and sequence complexity. Identifying repetitive DNA elements. Studying genome evolution across species. This process continues to be instrumental in advancing our knowledge of DNA structure and its biological implications.

5. Nucleic Acid Hybridization Nucleic acid hybridization is a molecular biology technique used to detect, identify, and quantify specific nucleic acid sequences. It involves annealing complementary single-stranded nucleic acids to form double-stranded hybrids through base pairing

6. Key Steps: Probe and Target Preparation : Probe: Single-stranded nucleic acid labeled with a detectable marker (e.g., radioactive isotope, fluorescent dye). Target: To analyze the nucleic acid sample (DNA or RNA) . 2 . Denaturation : Double-stranded nucleic acids are separated into single strands by heating or chemical treatment. 3 Hybridization : The probe and target are incubated under controlled conditions. Complementary sequences form hybrids through hydrogen bonding. 4. Detection and Analysis: Unbound probes are washed away. Hybridized probes are detected using fluorescence, autoradiography, or enzymatic assays.

7. Applications: Genetic mapping Paternity testing Forensics Population genetics Studying genetic diversity and diseases Although largely replaced by PCR and DNA sequencing, RFLP provides valuable insights into genetic variation in specific contexts.

8. Key Steps: 1. DNA Digestion: Genomic DNA is isolated and digested with restriction enzymes that cut at specific sequences, producing fragments of varying lengths. 2. Gel Electrophoresis: DNA fragments are separated by size on an agarose gel using an electric current. Smaller fragments migrate faster, creating distinct band patterns. 3 DNA Visualization: Fragments are visualized with stains like ethidium bromide or fluorescent dyes. 4. Analysis: Band patterns are compared to identify variations in fragment lengths (RFLPs). Differences arise from single nucleotide polymorphisms (SNPs) or insertions/deletions (indels) near restriction sites.

9. Steps Involved in Restriction Fragment Length Polymorphism (RFLP)

10. Amplified Fragment Length Polymorphism (AFLP ) AFLP is a molecular technique used to detect genetic variations across genomes without requiring prior sequence information. It is highly sensitive and reproducible, making it ideal for diverse applications . Key Steps: 1. DNA Digestion: Genomic DNA is isolated and digested with two restriction enzymes (frequent and less frequent cutters), producing fragments of varying lengths . 2. Adapter Ligation: Short DNA adapters are ligated to the fragment ends, serving as priming sites for PCR. 3. Preselective PCR: Initial PCR amplifies a subset of fragments using primers targeting the adapter sequences, reducing sample complexity . 4. Selective Amplification: PCR is performed with primers containing additional selective nucleotides, amplifying specific fragments based on sequence and size. 5. Fragment Analysis: Amplified fragments are separated by size using gel or capillary electrophoresis and visualized with fluorescent dyes or radioactive labels.

11. DNA Reassociation Nucleic Acid (NA) Hybridization Restriction Fragment Length Polymorphisms (RFLP) Amplified Fragment Length Polymorphisms (AFLP) Amplified DNA Ribosomal Restriction Analysis (ARDRA) Ribosomal Intergenic Spacer Analysis (RISA) Automated Ribosomal Intergenic Spacer Analysis (ARISA) Random Amplified Polymorphic DNA (RAPD) Stable Isotope Probing Ribotyping DNA Microarrays

12. Amplified Fragment Length Polymorphisms (AFLP ) AFLP is a molecular technique used to detect genetic variations across genomes without requiring prior sequence information. It is highly sensitive and reproducible, making it ideal for diverse applications. Key Steps: 1. DNA Digestion : Genomic DNA is isolated and digested with two restriction enzymes (frequent and less frequent cutters), producing fragments of varying lengths. 2. Adapter Ligation : Short DNA adapters are ligated to the fragment ends, serving as priming sites for PCR. 3. Preselective PCR : Initial PCR amplifies a subset of fragments using primers targeting the adapter sequences, reducing sample complexity. 4. Selective Amplification : PCR is performed with primers containing additional selective nucleotides, amplifying specific fragments based on sequence and size. 5. Fragment Analysis : Amplified fragments are separated by size using gel or capillary electrophoresis and visualized with fluorescent dyes or radioactive labels.

13. Applications: Genetic diversity studies Genetic mapping Plant breeding Population genetics Evolutionary biology AFLP generates high-resolution genetic fingerprints and identifies molecular markers associated with traits, providing valuable insights into genome variations .

14. Amplified Ribosomal DNA Restriction Analysis (ARDRA ) ARDRA is a molecular biology technique used to identify microbes and analyze microbial communities. It combines PCR amplification of ribosomal RNA (rRNA) genes with restriction enzyme digestion and gel electrophoresis to generate unique DNA fragment patterns Amplified Ribosomal DNA (rDNA) Restriction Analysis (ARDRA) Amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) is a modified method of  PCR   ribotyping . This simple and effective technique was first reported by Vaneechoutte and coworkers; they could distinguish nine of the 13 well-described taxa of the eubacterial family  Comamonadaceae  ( Vaneechoutte et al., 1992).

15. Steps in ARDRA: 1. PCR Amplification : Amplify a region of the rRNA gene, such as the 16S rRNA gene (bacteria) or 18S rRNA gene (fungi), using universal or group-specific primers. Target conserved regions flanking hypervariable regions for both conserved and variable sequence analysis. 2. Restriction Enzyme Digestion : Digest amplified DNA with restriction enzymes that recognize specific sequences within the rRNA gene. Generate DNA fragments of varying lengths, reflecting different microbial taxa or strains. 3. Gel Electrophoresis : Separate DNA fragments by size using agarose gel electrophoresis. Visualize fragments with ethidium bromide or fluorescent dyes and determine sizes relative to standards. 4. Analysis : Compare ARDRA profiles (fragment patterns) to evaluate microbial diversity and composition. Use clustering or principal component analysis (PCA) to group samples and assess community relationships. Sequence representative fragments for precise microbial identification

16. Applications: Microbial ecology and taxonomy Environmental microbiology Clinical microbiology (e.g., feces , saliva, sputum) Mirobial source tracking ARDRA is a cost-effective method for studying microbial diversity and dynamics in various environments and identifying indicators of environmental changes or diseases.

17. Rapid Imaging Spectrophotometric Array (RISA) RISA is a high-throughput technique used for analyzing DNA fragments from restriction enzyme digestion. It combines electrophoresis, imaging, and spectrophotometry to quickly and quantitatively assess DNA fragment sizes and their relative abundances. It is particularly useful in microbial ecology and environmental microbiology to study microbial community composition and diversity. Here's an overview of how the RISA technique works

18. Steps in the RISA Process : 1. DNA Fragment Generation : Environmental DNA is extracted from microbial communities in soil, water, or other environmental samples. The extracted DNA is subjected to restriction enzyme digestion, which cuts the DNA at specific recognition sites, producing a variety of DNA fragments of different sizes. 2. Electrophoresis: The digested DNA fragments are separated by size using gel electrophoresis. The DNA samples are loaded into an agarose gel, and an electric current is applied. DNA fragments migrate through the gel, with smaller fragments moving faster than larger ones, leading to size-based separation. 3. Imaging and Spectrophotometry : While the DNA fragments are migrating through the gel, they are imaged using a high-resolution system. The DNA is typically stained with a fluorescent dye (e.g., ethidium bromide or SYBR Green), which emits fluorescence when illuminated. A spectrophotometric detection system measures the fluorescence intensity of each fragment, providing quantitative data about fragment abundance. 4. Data Analysis : The imaging and fluorescence data are processed with specialized software. The software identifies individual DNA fragments based on their size and fluorescence intensity. A RISA profile is generated, which quantifies the diversity and composition of the microbial community by analyzing the DNA fragment sizes and their relative abundance.

19. Applications of RISA: Microbial Ecology : RISA provides high-resolution fingerprints of microbial communities, allowing researchers to study their diversity, composition, and changes over time. Environmental Monitoring : It is useful for assessing the impact of environmental factors (e.g., pollution, habitat change) on microbial communities. Biodiversity Studies : RISA can help evaluate the biodiversity of microbial communities in different environments. Ecosystem Management : It supports research on ecosystem function and management by monitoring microbial diversity and changes in response to environmental disturbances. Microbial Community Composition: RISA is valuable for identifying key taxa or functional groups within microbial communities, aiding in understanding microbial roles in ecosystems. RISA's rapid, sensitive, and quantitative approach makes it an important tool for ecological and environmental microbiology research.

20. Automated Ribosomal Intergenic Spacer Analysis (ARISA) ARISA is an advanced molecular biology technique used to profile microbial communities by analyzing the length variations in the ribosomal intergenic spacer (ITS) regions. It is an automated extension of the traditional Ribosomal Intergenic Spacer Analysis (RISA), utilizing capillary electrophoresis for fragment analysis. ARISA allows for high-throughput and sensitive assessment of microbial community composition and diversity. Automated ribosomal intergenic spacer analysis is a new method of testing for bacterial strains. It is now being applied to cheese. ARISA has allowed for increased accuracy in the area of cheese defects and gas formers .

21. Steps in the ARISA Process: 1. DNA Extraction : DNA is extracted from environmental samples (soil, water, etc.) using standard molecular biology methods, focusing on microbial communities. 2. PCR Amplification : The ribosomal intergenic spacer (ITS) regions, located between the 16S and 23S rRNA genes, are amplified using universal primers that target conserved regions flanking the ITS regions. The PCR primers are fluorescently labeled, enabling easy detection of the amplified fragments during the analysis. 3. Fragment Analysis : The fluorescently labeled PCR products are injected into a capillary filled with a polymer matrix and subjected to an electric field (capillary electrophoresis). As the DNA fragments migrate through the capillary, they are detected by a fluorescence detector, which measures their size and intensity based on the fluorescent signal. 4 . Data Analysis : The fragment analysis data are processed using specialized software to identify and quantify DNA fragments based on their size and fluorescence intensity. The resulting ARISA profiles provide a quantitative assessment of the microbial community composition and diversity

22. Random Amplified Polymorphic DNA (RAPD) RAPD is a PCR-based technique used to detect genetic variation among individuals or populations. It amplifies random segments of genomic DNA, generating unique DNA fingerprints without the need for prior sequence information. RAPD is simple, cost-effective, and provides valuable insights into genetic diversity.

23. Steps in the RAPD Process: 1. Primer Selection : RAPD uses short, random primers (10-20 nucleotides long) that can bind to multiple genomic sites. These primers are designed with high degeneracy to enhance the likelihood of annealing to different genomic loci. 2. DNA Amplification : Genomic DNA is extracted from the samples to be analyzed. The DNA is then amplified using a single random primer at a low annealing temperature, which allows primers to bind to multiple sites, generating DNA fragments of various sizes. 3. Gel Electrophoresis : The PCR products are separated by size using gel electrophoresis. DNA fragments move through the agarose gel under an electric field, with smaller fragments migrating faster than larger ones. 4. Visualization: After electrophoresis, the DNA bands are stained with ethidium bromide or fluorescent dyes. The bands are visualized under UV light, where each band corresponds to a distinct DNA fragment. 5. Analysis : The RAPD profiles, consisting of bands of various sizes, are analyzed to assess genetic variation. Similarity analysis, such as clustering algorithms or principal component analysis (PCA), can be used to compare genetic diversity and relationships among samples.

24. Applications of RAPD: Genetics and Genomics : RAPD is used to detect genetic variation within populations, identify molecular markers and understand genetic diversity. Plant Breeding : It helps in selecting desirable traits in crops and assessing genetic improvement. Population Genetics : RAPD is used to study genetic relationships within and between populations, revealing information about population structure and genetic drift. Biodiversity Studies : RAPD is applied in biodiversity conservation and studies of genetic diversity in various species. Ecological Research : It provides insights into the genetic differentiation of populations in natural environments.

25. Stable Isotope Probing (SIP) Stable isotope probing (SIP) is a technique used in microbial ecology to identify and characterize active microbial populations involved in specific metabolic processes by tracking the incorporation of stable isotopes like carbon-13 (^13C) or nitrogen-15 (^15N) into microbial biomass. It provides insights into microbial identity, metabolic activity, and the contribution of different microbial groups to ecosystem processes

26. Steps Involved in SIP : 1. Isotope Labeling : Microorganisms are incubated with a substrate labeled with a stable isotope, such as ^13C-glucose (carbon source) or ^15N-ammonium (nitrogen source). The microbial communities incorporate the labeled substrate into their biomass during metabolism, incorporating the stable isotope into biomolecules like DNA, RNA, proteins, and lipids. 2. Density Gradient Centrifugation : After incubation, microbial cells are separated by their density using a density gradient medium (e.g., cesium chloride or sucrose) and subjected to ultracentrifugation. This step separates microbial biomass into fractions based on their buoyant density, allowing the isotopically labeled biomass to be isolated. 3. Isotope Analysis : The fractions containing labeled biomass are collected and subjected to molecular techniques such as DNA or RNA extraction. Isotopically labeled nucleic acids are separated from unlabeled ones using methods like isopycnic centrifugation or gradient fractionation. The labeled nucleic acids are then analyzed using sequencing or PCR-based techniques to identify active microbial populations involved in metabolizing the labeled substrate. 4. Data Interpretation : The resulting data help link microbial identity with their metabolic function, shedding light on the role of specific microbial groups in biogeochemical cycles like carbon and nitrogen cycling. This allows for the quantification of microbial contributions to ecosystem functions and provides a deeper understanding of microbial community dynamics and functional diversity .

27. Ribotyping Ribotyping is a molecular biology technique used to characterize bacterial strains based on the patterns of ribosomal RNA (rRNA) genes. This method is primarily used for bacterial strain typing, taxonomic classification, epidemiological studies, and microbial diversity analysis. Ribotyping helps in identifying the genetic relatedness of bacterial strains by analyzing the rRNA gene operons through restriction enzyme digestion.

28. Steps Involved in Ribotyping: 1. DNA Extraction : Genomic DNA is extracted from bacterial cultures or clinical samples using standard molecular biology methods. 2 . Restriction Enzyme Digestion : The extracted DNA is digested with one or more restriction enzymes. These enzymes recognize specific DNA sequences within the rRNA gene operon. The restriction enzymes cleave the DNA at these recognition sites, producing a set of DNA fragments of varying lengths. 3. Gel Electrophoresis : The digested DNA fragments are separated by size through gel electrophoresis. DNA fragments are loaded onto an agarose gel, and an electric current is applied. The DNA migrates through the gel matrix, with smaller fragments moving more quickly than larger ones.

29. 4. Transfer and Hybridization : After electrophoresis, the DNA fragments are transferred onto a membrane (nylon or nitrocellulose). The membrane-bound DNA is hybridized with a labeled probe that targets the conserved regions of the rRNA genes. 5 . Detection and Analysis : After washing the membrane to remove any unbound probe, the hybridized DNA fragments are visualized using autoradiography, chemiluminescence, or fluorescence. The resulting ribotype is a unique banding pattern that reflects the distribution of rRNA gene fragments in the bacterial genome. These patterns are compared across bacterial strains or isolates to assess genetic relatedness and identify phylogenetic relationships

30. Applications of Ribotyping: Baterial Strain Typing : Ribotyping helps distinguish between bacterial strains, providing insights into their genetic diversity and relationship. Epidemiological Surveillance : This technique is useful for tracking bacterial pathogens, investigating outbreaks of infectious diseases, and determining the source of infections. Microbial Diversity Analysis : Ribotyping can be used to study the diversity of microbial populations in environmental samples, such as soil, water, and food. Taxonomic Classification : It aids in the classification and phylogenetic analysis of bacterial species by providing a genetic fingerprint based on rRNA genes. Evolutionary Relationships : Ribotyping provides insights into the evolutionary dynamics of bacterial species, helping to trace the genetic lineage and adaptation of pathogens

31. Advantages of Ribotyping : • High Reproducibility : Ribotyping produces highly reproducible results, making it an effective tool for strain comparison and surveillance. • Effective for Pathogen Tracking: It helps track the spread of bacterial pathogens in clinical settings, food safety, and environmental monitoring. • No Need for Full Genome Sequencing: Unlike whole-genome sequencing, ribotyping can provide detailed genetic information without the need for complete genome sequencing.

32. DNA Microarrays DNA microarrays, also known as DNA chips or gene chips, are powerful tools in genomics and molecular biology, used to analyze the expression levels of thousands of genes or nucleic acid sequences simultaneously in a single experiment. They consist of a solid support, usually a glass slide or silicon wafer, onto which thousands to millions of nucleic acid probes are attached in a defined array format .

33. DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached in an organized, pre-determined grid fashion. Each spot of DNA, called a probe, represents a single gene. DNA microarrays can analyze the expression of tens of thousands of genes simultaneously. There are several synonyms for DNA microarrays, such as DNA chips, gene chips, DNA arrays, gene arrays, and biochips. Types of DNA Microarrays DNA microarrays, also known as DNA chips, can be categorized into two main types based on the probes used for hybridization: 1 . cDNA-based Microarray (Spotted DNA Arrays or cDNA Arrays) 2. Oligonucleotide-based Microarray (Gene Chips ) 1. cDNA-based Microarray (Spotted DNA Arrays or cDNA Arrays) These microarrays are prepared using cDNA, which is complementary DNA synthesized from mRNA. The main features of this type are:

34. Preparation: cDNA Amplification: The cDNAs are amplified using PCR (Polymerase Chain Reaction) to ensure there is enough material for the array. Spotting: The amplified cDNAs are immobilized on a solid support (e.g., a nylon filter or a glass slide), usually 1 x 3 inches in size. This process is done through capillary action or using mechanical/robotic methods for precise delivery. Probe DNA: The cDNAs serve as probe DNA. The immobilization process involves placing small volumes of cDNA onto the array surface, ensuring physical contact for hybridization. Applications: cDNA microarrays are widely used to study gene expression by comparing mRNA levels in different samples, such as comparing normal vs. diseased tissue or response to different treatments.

35. Comparison of cDNA and Oligonucleotide Microarrays Feature cDNA Microarrays (Spotted DNA Arrays) Oligonucleotide Microarrays (Gene Chips) Probe Type cDNA derived from mRNA Short DNA oligonucleotides (20-25 nucleotides) Probe Representation Single representation per gene Multiple probes for each gene Fabrication Method PCR amplification and spotting Photolithography Customization Can be custom designed for specific genes Typically off-the-shelf, standardized arrays Detection of Variations Less precise for detecting SNPs and small variations Highly precise for detecting SNPs and variations Applications Gene expression analysis Gene expression, SNP detection, genotyping

36. Applications of DNA Microarrays : Gene Expression Analysis : DNA microarrays are widely used to study the expression of genes across various conditions, helping to understand biological processes, disease mechanisms, and cellular responses to environmental stimuli. Genetic Variation : Microarrays help in detecting sequence variants, such as mutations or SNPs, to study genetic diversity, disease susceptibility, and evolutionary relationships. Epigenetics: DNA microarrays are also used to study epigenetic modifications such as DNA methylation and histone modifications, which play crucial roles in gene regulation and disease development. Drug Discovery : They are used in pharmaceutical research to discover potential drug targets, screen for therapeutic compounds, and analyze the effects of drug treatments on gene expression. Personalized Medicine : Microarrays play a vital role in identifying genetic markers and expression profiles to tailor individualized treatment plans based on a person's genetic makeup. Agriculture: DNA microarrays are employed in agriculture for plant breeding, pest resistance studies, and analyzing gene expression related to plant growth, disease resistance, and crop yields.

37. Methodology: 1. Sample Collection & DNA Extraction: Environmental samples (soil, water, etc.) are collected, and total DNA is extracted. 2. DNA Sequencing: High-throughput sequencing (e.g., NGS) generates millions of DNA sequences, targeting specific genes or whole genomes. 3. Data Analysis: Bioinformatics tools are used to identify microbial taxa, genes, and metabolic pathways. Sequences are clustered into Operational Taxonomic Units (OTUs) or Amplicon Sequence Variants (ASVs). 4. Taxonomic & Functional Profiling: Taxonomic profiling reveals the diversity of microbes, while functional profiling identifies genes involved in processes like metabolism and biodegradation. 5. Comparative Analysis: Data from different samples are compared to uncover patterns and ecological relationships.