Class-Tm Cot ValuePARADOX MOLECULAR BIOLOGY .pptx

FOR MOLECULAR BIOLOGY DEPTH

1. C-value paradox *Factors Contributing to the C-value Paradox*  1. *Non-coding DNA*: Much of the DNA in eukaryotic genomes does not code for proteins, and its function is not fully understood. 2. *Gene duplication*: Gene duplication events can increase genome size without necessarily adding new functional genes. 3. *Transposable elements*: Mobile genetic elements can replicate and accumulate, contributing to increased genome size. 4. *Evolutionary rate variation*: Different species evolve at different rates, influencing genome size. *C-value Paradox Aspects* 1. Causes (transposable elements, gene duplication) 2. Implications (evolutionary complexity) 3. Theories (neutral, selfish DNA, junk DNA) C-value is the amount of DNA in one haploid set of chromosomes. The C-value paradox arises from the fact that  different organisms having the same general level of complexity and even organisms belonging to the same genus often have widely different C-values . 'C-value' means  the 'constant' (or 'characteristic') value of haploid DNA content per nucleus , typically measured in picograms (1 picogram is roughly 1 gigabase ). *Relevant Fields* 1. Comparative genomics 2. Functional genomics 3. Epigenomics 4. Synthetic biology

2. Schematic  karyogram  of a human. It shows 22  homologous chromosomes , both the female (XX) and male (XY) versions of the  sex chromosome  (bottom right), as well as the  mitochondrial genome  (to scale at bottom left). The blue scale to the left of each chromosome pair (and the mitochondrial genome) shows its length in terms of millions of DNA  base pairs .

3. Conversion from picograms ( pg ) to base pairs (bp)

4. *Examples of the C-value Paradox* 1. *Human genome*: Approximately 3.2 billion base pairs, with only about 2% coding for proteins. 2. *Lamprey genome*: Despite being a relatively simple organism, its genome is larger than that of humans. 3. *Salmon genome*: Has a larger genome than humans, despite being a less complex organism. 4. *Onion genome*: Has a larger genome than humans, with approximately 5 times more DNA.

5. *Causes* 1. *Transposable elements*: Mobile genetic elements replicating and accumulating. 2. *Gene duplication*: Increased gene copy numbers. 3. *Non-coding DNA*: Non-functional or regulatory sequences. 4. *Evolutionary rate variation*: Different species evolving at different rates. *Characteristics*  1. *Genome size variability*: Vast differences in DNA content among species. 2. *Lack of correlation*: No direct link between genome size and organism complexity. 3. *Non-coding DNA dominance*: Majority of DNA doesn't code for proteins.

6. *Structure* (HUMAN) 1. *3.2 billion base pairs*: Total DNA length. 2. *23 pairs of chromosomes*: 22 autosomal, 1 sex chromosome. 3. *20,000-25,000 protein-coding genes*: Only 2% of genome. 4. *Non-coding DNA*: 98% of genome, regulating gene expression. *Composition* 1. *A (Adenine): 29.3%* 2. *T (Thymine): 29.3%* 3. *G (Guanine): 20.7%* 4. *C (Cytosine): 20.7%* *Functions* 1. *Protein-coding genes*: Encode proteins. 2. *Non-coding RNA genes*: Regulate gene expression. 3. *Regulatory elements*: Control gene expression. 4. *Junk DNA*: Unknown functions.

7. *Interesting Facts* 1. *99.9% similarity*: Humans share identical DNA. 2. *8% viral DNA*: Integrated viral sequences. 3. *1,500-2,000 genetic disorders*: Caused by genetic mutations. 4. *23andMe*: Popular genetic testing platform. *Genetic Variation* 1. *Single Nucleotide Polymorphisms (SNPs)*: Common variations. 2. *Copy Number Variations (CNVs)*: Gene duplication/deletion. 3. *Insertions/Deletions (Indels)*: Small genetic changes. *Applications* 1. *Personalized medicine*: Tailored treatments. 2. *Genetic counseling*: Informed reproductive decisions. 3. *Forensic analysis*: DNA identification. 4. *Gene therapy*: Treating genetic disorders. If all the DNA in your body was put end to end, it would reach to the sun and back over 600 times  (100 trillion times six feet divided by 92 million miles). It would take a person typing 60 words per minute, eight hours a day, around 50 years to type the human genome.

8. *Genetic Testing* 1. *Diagnostic Testing*: Identifying genetic disorders. 2. *Predictive Testing*: Determining disease risk. 3. *Carrier Testing*: Identifying genetic carriers. 4. *Prenatal Testing*: Testing fetal genetic health. *Gene Therapy* 1. *Somatic Gene Therapy*: Treating non-reproductive cells. 2. *Germline Gene Therapy*: Altering reproductive cells. 3. *Gene Editing*: CRISPR/Cas9 technology. Ethical Considerations* 1. *Genetic Privacy*: Protecting genetic information. 2. *Informed Consent*: Ensuring understanding of genetic testing. 3. *Genetic Discrimination*: Preventing unfair treatment. 4. *Gene Patenting*: Regulating genetic intellectual property.

9. *Smallest Genomes* 1. *Viroid*: 246-467 base pairs (bp) 2. *Circovirus*: 2,000 bp 3. *Bacteriophage MS2*: 3,569 bp 4. *Mycoplasma genitalium *: 580,000 bp (smallest bacterial genome) *Largest Genomes* 1. *Polyploid plants (Paris japonica)*: 149 billion bp 2. *Lamprey*: 138 billion bp 3. *Salmon*: 63 billion bp 4. *Human*: 3.2 billion bp *Comparative Genome Sizes* 1. *E. coli*: 4.6 million bp 2. *Yeast*: 12 million bp 3. *Fruit fly*: 180 million bp 4. *Wheat*: 17 billion bp 5. *Onion*: 15.8 billion bp *Factors Influencing Genome Length* 1. Polyploidy 2. Gene duplication 3. Transposable elements 4. Evolutionary pressures

10. Melting temperature (Tm), buoyant density, and DNA reassociation kinetics (Cot curve analysis): *Melting Temperature (Tm)* 1. *Definition*: Temperature at which 50% of DNA double strands separate into single strands. 2. *Factors influencing Tm*: GC content, salt concentration, pH, and DNA length. 3. *Applications*: DNA hybridization, PCR, DNA sequencing.

11. *DNA Melting Temperature (Tm)* 1. *Calculation methods*: Online Tm calculators (e.g., IDT, Tm Calculator) 2. *Factors influencing Tm*: GC content, salt concentration, pH, DNA length 3. *Applications*: DNA hybridization, PCR, DNA sequencing 4. *Measurement methods*: UV spectrophotometry, fluorescence

12. Caesium chloride ( CsCl ) solution and two morphological types of  rotavirus . Following centrifugation at 100g a density gradient forms in the CsCl solution and the virus particle separate according to their densities. Buoyant density centrifugation  (also  isopycnic centrifugation  or  equilibrium density-gradient centrifugation ) uses the concept of  buoyancy  to separate molecules in solution by their differences in density.

13. *Buoyant Density* 1. * CsCl density gradient centrifugation*: Separating DNA by buoyant density *Principle*: DNA molecules separate based on density differences. 2. *Applications*: DNA purification, genome analysis 3. *Measurement methods*: Density gradient centrifugation, ultracentrifugation 4. *Resources*: NCBI, Scopus, PubMed

14. *DNA Reassociation Kinetics (Cot Curve Analysis)* 1. *Definition*: Measures DNA reannealing rate after denaturation. 2. *Cot value*: Product of DNA concentration (C) and incubation time (t). . *Measurement methods*: Spectrophotometry, fluorescence *Cot curve phases*: Rapid (simple sequences), intermediate (moderately repetitive), slow (complex/unique sequences) 3. *Cot curve*: Plot of reassociation rate vs. Cot value. 4. *Applications*: Genome complexity, repetitive sequence analysis. Repetitive DNA sequences renature at lower C t values than single-copy sequences. C t analysis , a technique based on the principles of  DNA reassociation kinetics , is a biochemical technique that measures how much  repetitive DNA  is in a DNA sample such as a  genome . [1]  It is used to study  genome  structure and organization and has also been used to simplify the sequencing of genomes that contain large amounts of repetitive sequence.

15. *Key Protocols and Techniques* 1. *Thermal denaturation*: Heating DNA to measure Tm 2. * CsCl density gradient centrifugation*: Separating DNA by buoyant density 3. *DNA reassociation kinetics*: Measuring Cot curves 4. *PCR*: Amplifying specific DNA sequences *Online Resources* 1. National Center for Biotechnology Information (NCBI) 2. Scopus 3. PubMed 4. Biochemistry textbooks (e.g., Alberts et al., Campbell et al.) 5. Online Tm calculators (e.g., IDT, Tm Calculator) 6. DNA analysis software (e.g., BLAST, GenBank) *Research Articles* 1. "DNA Melting Temperature" (Journal of Molecular Biology) 2. "Buoyant Density of DNA" (Biophysical Journal) 3. "Cot Curve Analysis" (Methods in Enzymology)

16. Types of DNAs •Single copy DNA is a specific  DNA sequence  that is present only once in the genome. • Repetitive DNA  is a DNA segment with a specific DNA sequence that is repeated multiple times in the genome. •Moderately repetitive DNA refers to 10–105 copies of the sequence per genome. •Moderate repeated DNA is found primarily in noncoding sequences. •Highly repetitive DNA describes DNA sequence present in greater than 105 copies per genome. •Highly repeated DNA is found primarily in  centromere  and  telomere  regions as  tandem repeats . •NA has two DNA chains; one is oriented 5′.

17. Composition of the human genome

18. Genome sizes and corresponding composition of six major model organisms as pie charts. The increase in  genome  size correlates with the vast expansion of noncoding (i.e., intronic, intergenic, and interspersed repeat sequences) and repeat  DNA  (e.g., satellite, LINEs,  short interspersed nuclear element  (SINEs), DNA ( Alu sequence ), in red) sequences in more complex multicellular organisms. This expansion is accompanied by an increase in the number of  epigenetic  mechanisms (particularly repressive) that regulate the genome. Expansion of the genome also correlates with an increase in size and complexity of transcription units, except for plants. P =  Promoter  DNA element.

19. *Levels of Genomic Organization:* 1. Chromosomal level: arrangement of chromosomes in the nucleus 2. Chromatin level: structure of chromatin (DNA + histones) 3. Gene level: organization of genes within chromatin 4. Regulatory level: arrangement of regulatory elements (enhancers, promoters) *Chromosomal Organization:* 1. Chromosome structure: centromere, telomeres, and chromosomal arms 2. Chromosome territories: spatial arrangement in the nucleus 3. Chromosome interactions: inter-chromosomal interactions *Chromatin Organization:* 1. Nucleosome structure: DNA wrapped around histone octamer 2. Chromatin remodeling : dynamic changes in chromatin structure 3. Histone modifications: epigenetic marks (methylation, acetylation) *Gene Organization:* 1. Gene structure: exons, introns, and regulatory elements 2. Gene expression: transcriptional regulation 3. Gene clusters: coordinated regulation of gene expression *Regulatory Elements:* 1. Promoters: initiate transcription 2. Enhancers: increase transcription 3. Silencers: decrease transcription 4. Insulators: regulate chromatin interactions

20. *Types of Repetitive DNA:* 1. Satellite DNA: Short sequences (5-100 bp) repeated in tandem. 2. Minisatellites: Longer sequences (100-500 bp) repeated in tandem. 3. Microsatellites: Short sequences (1-5 bp) repeated in tandem. 4. Transposable elements (TEs): Mobile DNA sequences. 5. Retrotransposons: TEs that replicate via RNA intermediates. 6. DNA transposons: TEs that move directly from one location to another. *Functions of Repetitive DNA:* 1. Chromosomal organization and structure. 2. Gene regulation and expression. 3. Genome stability and repair. 4. Evolutionary diversity. 5. Epigenetic regulation. *Methods for Analyzing Repetitive DNA:* 1. Southern blotting. 2. PCR (Polymerase Chain Reaction). 3. In situ hybridization. 4. Fluorescence in situ hybridization (FISH). 5. Next-generation sequencing (NGS). 6. Bioinformatics tools (e.g., RepeatMasker , RepeatExplorer ).

21. *1. Satellite DNA* - Short sequences (5-100 bp) repeated in tandem - Found in centromeres, telomeres, and heterochromatic regions - Examples: human alpha-satellite, mouse minor satellite *2. Minisatellites* - Longer sequences (100-500 bp) repeated in tandem - Found in subtelomeric regions and gene-rich areas - Examples: human minisatellite, mouse DAF161 *3. Microsatellites* - Short sequences (1-5 bp) repeated in tandem - Found throughout the genome, including gene-rich areas - Examples: CA repeats, AT repeats  *4. Transposable Elements (TEs)* - Mobile DNA sequences that can jump to new locations - Classified into two main types:     - DNA transposons (e.g., mariner, Tc1)     - Retrotransposons (e.g., LINEs, SINEs)  *5. Retrotransposons* - Long Interspersed Nuclear Elements (LINEs) - Short Interspersed Nuclear Elements (SINEs) - Long Terminal Repeats (LTRs)

22. *6. DNA Transposons* - Mariner-like elements - Tc1-like elements - hAT -like elements  *7. Tandem Repeats* - Sequences repeated in tandem, often found in gene-poor regions - Examples: human beta-globin pseudogene, mouse major satellite  *8. Interspersed Repeats* - Sequences dispersed throughout the genome - Examples: Alu repeats, SINEs  *9. Long Range Repeats* - Sequences repeated over long distances (>100 kb) - Examples: human olfactory receptor gene clusters  *10. Segmental Duplications* - Large-scale duplications of genomic segments - Examples: human chromosome 2, mouse chromosome 1

23. *Databases for Repetitive DNA:* 1. Repbase . 2. RepeatMasker . 3. UCSC Genome Browser. 4. ENSEMBL Genome Browser. 5. NCBI Genome Browser. *Diseases Associated with Repetitive DNA:* 1. Fragile X syndrome. 2. Huntington's disease. 3. Myotonic dystrophy. 4. Spinocerebellar ataxia. 5. Cancer. *Evolutionary Significance:* 1. Genome evolution. 2. Speciation. 3. Genetic diversity. 4. Adaptation to environments. 5. Evolution of gene regulation.