Biology Notes of Molecular Basis of Inheritance CBSE Class 12

This chapter explores the molecular basis of inheritance, focusing on DNA as the primary genetic material in most organisms, building upon the understanding of inheritance patterns. It details the structure of DNA , how it is packaged within cells, and the historical search that confirmed its role as the genetic material through experiments like Griffith's transformation principle and the Hershey-Chase experiment. Key processes such as DNA replication (semiconservative), transcription (DNA to RNA), and translation (RNA to protein) are explained, along with the concept of the genetic code and how gene expression is regulated. The Human Genome Project (HGP), its goals, and findings are discussed, as is DNA fingerprinting and its applications based on DNA polymorphism .

1. The DNA

• Nucleic Acids: Polymers of nucleotides.
• Deoxyribonucleic acid ( DNA ).
• Ribonucleic acid ( RNA ).
• Role of DNA: Acts as the genetic material in most organisms.
• Role of RNA: Acts as genetic material in some viruses. Mostly functions as a messenger. Has additional roles as adapter, structural, and catalytic molecule.
• DNA Structure: A long polymer of deoxyribonucleotides .
• Length defined by number of nucleotides or base pairs (bp).
• Example lengths: Bacteriophage φ×174 (5386 nucleotides), Bacteriophage lambda (48502 bp), Escherichia coli (4.6 × 10⁶ bp), human haploid content (3.3 × 10⁹ bp).

2. Structure of Polynucleotide Chain

• Nucleotide Components:
1. Nitrogenous base.
2. Pentose sugar (ribose for RNA, deoxyribose for DNA).
3. Phosphate group.
• Nitrogenous Bases:
• Purines: Adenine (A), Guanine (G).
• Pyrimidines: Cytosine (C), Uracil (U), Thymine (T).
• Cytosine (C) is common to both DNA and RNA.
• Thymine (T) is present in DNA.
• Uracil (U) is present in RNA, replacing Thymine.
• Nucleoside Formation: Nitrogenous base linked to 1' C of pentose sugar via an N-glycosidic linkage .
• Examples: Adenosine/deoxyadenosine, Guanosine/deoxyguanosine, Cytidine/deoxycytidine, Uridine/deoxythymidine.
• Nucleotide Formation: Phosphate group linked to 5' C of a nucleoside via a phosphoester linkage .
• Polynucleotide Chain Formation: Nucleotides linked by 3'-5' phosphodiester linkage .
• Polarity: A polymer has a free phosphate at the 5'-end of the sugar (5'-end) and a free OH at the 3' C of the sugar (3'-end).
• Backbone: Formed by sugar and phosphates. Nitrogenous bases project from the backbone.
• RNA Specific Differences:
• Additional –OH group at 2'-position in the ribose of every nucleotide residue.
• Uracil (U) instead of Thymine (T).

3. The Double Helix Model

• Discovery: DNA as an acidic substance ("Nuclein") first identified by Friedrich Meischer in 1869.
• Model Proposal: James Watson and Francis Crick proposed the Double Helix model in 1953.
• Basis: Based on X-ray diffraction data (Maurice Wilkins and Rosalind Franklin) and Erwin Chargaff's rules.
• Chargaff's Rules: In double-stranded DNA, the ratios of Adenine to Thymine (A:T) and Guanine to Cytosine (G:C) are constant and equal to one.
• Salient Features of DNA Double Helix:
• Made of two polynucleotide chains .
• Backbone: Sugar-phosphate. Bases project inside.
• Chains have anti-parallel polarity (one 5'→3', the other 3'→5').
• Bases pair through hydrogen bonds (H-bonds) .
• Adenine (A) forms two H-bonds with Thymine (T).
• Guanine (G) forms three H-bonds with Cytosine (C).
• A purine always pairs with a pyrimidine, creating approximately uniform distance between strands.
• Chains coil in a right-handed fashion .
• Pitch of helix: 3.4 nm.
• Roughly 10 bp per turn .
• Distance between bp: Approximately 0.34 nm.
• Plane of one base pair stacks over the other, conferring stability along with H-bonds.
• Complementarity: Base pairing makes the two strands complementary. Sequence of one strand predicts the other.
• Genetic Implication: Complementarity allows each strand to act as a template for a new strand, resulting in two identical daughter DNA molecules during replication. This explained the genetic implications of the structure.

4. Central Dogma

• Proposed by Francis Crick.
• States that genetic information flows from DNA → RNA → Protein .
• In some viruses, flow is reversed: RNA → DNA .

5. Packaging of DNA Helix

• DNA is very long (e.g., ~2.2 metres in a typical mammalian cell), much larger than the nucleus (~10⁻⁶ m).
• Prokaryotes (e.g., E. coli ) :
• No defined nucleus.
• DNA is held with positively charged proteins in a region called the nucleoid .
• DNA in the nucleoid is organized in large loops held by proteins.
• Eukaryotes : More complex organization.
• Involves positively charged, basic proteins called histones .
• Histones are rich in basic amino acids (lysine, arginine) which carry positive charges.
• Histones organize into a unit of eight molecules called a histone octamer .
• Negatively charged DNA wraps around the positively charged histone octamer to form a nucleosome .
• A typical nucleosome contains 200 bp of DNA.
• Nucleosomes are the repeating unit of chromatin in the nucleus.
• Chromatin appears as " beads-on-string " structure under electron microscope.
• Beads-on-string structure is further packaged into chromatin fibers .
• Chromatin fibers coil and condense to form chromosomes during cell division.
• Higher-level packaging requires additional proteins: Non-histone Chromosomal (NHC) proteins .
• Chromatin Structure and Activity:
• Loosely packed chromatin: Euchromatin (stains light, transcriptionally active).
• More densely packed chromatin: Heterochromatin (stains dark, transcriptionally inactive).

6. The Search for Genetic Material

• Quest for genetic inheritance mechanism reached molecular level by 1926. Chromosomes identified as likely location, but the molecule wasn't known.
• Genetic material was initially thought to be protein.

Transforming Principle (Frederick Griffith, 1928):

• Experiments with Streptococcus pneumoniae (causes pneumonia).
• S strain (virulent, smooth coat) killed mice.
• R strain (non-virulent, rough coat) did not kill mice.
• Heat-killed S strain did not kill mice.
• Mixture of heat-killed S and live R strain killed mice.
• Living S bacteria recovered from dead mice.
• Conclusion: R strain was transformed by heat-killed S strain. A " transforming principle " from S strain enabled R strain to synthesize the smooth coat and become virulent. Suggested transfer of genetic material, but biochemical nature undefined.

Biochemical Characterisation of Transforming Principle (Oswald Avery, Colin MacLeod, Maclyn McCarty, 1933-44):

• Purified biochemicals (proteins, DNA, RNA) from heat-killed S cells.
• Found that DNA alone from S bacteria caused R bacteria to become transformed.
• Protease (protein-digesting) and RNase (RNA-digesting) enzymes did not affect transformation.
• DNase (DNA-digesting) inhibited transformation.
• Conclusion: DNA is the hereditary material . Not all biologists were convinced initially.

The Genetic Material is DNA (Alfred Hershey and Martha Chase, 1952):

• Unequivocal proof that DNA is the genetic material.
• Worked with bacteriophages (viruses that infect bacteria).
• Bacteriophages attach to bacteria and inject genetic material.
• Experiment to determine if protein or DNA entered bacteria.
• Grew viruses with radioactive phosphorus ( ³²P ) - labels DNA (DNA has P, protein doesn't).
• Grew viruses with radioactive sulfur ( ³⁵S ) - labels protein (protein has S, DNA doesn't).
• Allowed radioactive phages to infect E. coli .
• Agitated mixture in a blender to remove viral coats.
• Centrifuged to separate viruses from bacteria.
• Results:
• Bacteria infected with ³²P-labeled DNA were radioactive (DNA entered bacteria).
• Bacteria infected with ³⁵S-labeled protein were not radioactive (protein did not enter bacteria).
• Conclusion: DNA is the genetic material passed from virus to bacteria.

7. Properties of Genetic Material (DNA versus RNA)

• Although DNA is the predominant genetic material, RNA serves as genetic material in some viruses.
• RNA performs dynamic functions (messenger, adapter).
• A molecule acting as genetic material must fulfill criteria:
1. Replication: Ability to generate replicas. Both DNA and RNA can direct duplication based on base pairing/complementarity. Proteins cannot fulfill this.
2. Stability: Chemically and structurally stable.
• Stability evident in Griffith's heat-killed S strain experiment. Heat separated DNA strands, but they could re-associate.
• RNA has a reactive 2'-OH group in ribose, making it labile and easily degradable. RNA is also catalytic, hence reactive.
• DNA is chemically less reactive and structurally more stable than RNA .
• Presence of Thymine (T) in DNA (vs Uracil in RNA) also confers additional stability.
3. Mutation: Provide scope for slow changes (mutation) for evolution. Both DNA and RNA can mutate. RNA mutates faster due to instability. RNA viruses evolve faster.
4. Expression: Ability to express itself ("Mendelian Characters"). RNA can directly code for protein synthesis and express characters. DNA is dependent on RNA for protein synthesis. Protein synthesis machinery evolved around RNA.
• Conclusion: Both can function as genetic material. DNA is preferred for storage of genetic information due to greater stability . RNA is better for transmission of genetic information.

8. RNA World

• Evidence suggests RNA was the first genetic material .
• Essential life processes (metabolism, translation, splicing) evolved around RNA.
• RNA acted as genetic material and catalysts (ribozymes).
• RNA was reactive and unstable due to catalytic nature.
• DNA evolved from RNA through chemical modifications making it more stable.
• Double-stranded DNA with complementary strands resists changes by evolving repair processes.

9. Replication

• Process of copying DNA.

Semiconservative DNA Replication:

• Proposed by Watson and Crick.
• The two strands separate and each acts as a template for new complementary strand synthesis.
• Each new DNA molecule has one parental and one newly synthesised strand.

Experimental Proof (Matthew Meselson and Franklin Stahl, 1958):

• Grew E. coli in medium with heavy nitrogen isotope ¹⁵N for many generations (DNA becomes heavy).
• Transferred cells to medium with normal ¹⁴N. Took samples at intervals.
• Extracted DNA and measured density using CsCl density gradient centrifugation.
• Results:
• After 1 generation (20 minutes): DNA had hybrid/intermediate density . (One strand ¹⁵N, one ¹⁴N).
• After 2 generations (40 minutes): DNA was composed of equal amounts of hybrid DNA and 'light' DNA (both strands ¹⁴N).
• Similar experiments with radioactive thymidine on Vicia faba by Taylor and colleagues also proved semiconservative replication in chromosomes.

10. Machinery and Enzymes of Replication

• Process requires a set of catalysts (enzymes).
• Main Enzyme: DNA-dependent DNA polymerase.
• Uses a DNA template to catalyze polymerisation of deoxynucleotides.
• Highly efficient (~2000 bp per second in E. coli ) and accurate (mistakes lead to mutations).
• Polymerisation direction: Only 5' → 3' .
• Substrates & Energy: Deoxyribonucleoside triphosphates. Serve as substrates and provide energy (high-energy phosphates).
• Replication Fork: Replication occurs within a small opening of the DNA helix called a replication fork. DNA strands cannot be separated along the entire length due to high energy requirement.
• Synthesis at Replication Fork:
• Template strand 3'→5': Continuous replication .
• Template strand 5'→3': Discontinuous replication . Synthesized as fragments.
• DNA Ligase: Joins the discontinuously synthesised fragments.
• Initiation: DNA polymerases cannot initiate replication on their own.
• Replication starts at definite regions called origin of replication .
• Requirement of origin of replication is why vectors are needed in recombinant DNA procedures.
• Eukaryotes: Replication occurs during the S-phase of the cell cycle. Replication and cell division must be highly coordinated (failure can lead to polyploidy).

11. Transcription

• Process of copying genetic information from one strand of DNA into RNA .
• Principle of complementarity applies, but Adenosine (A) pairs with Uracil (U) instead of Thymine (T).
• Unlike replication (whole DNA copied), only a segment of DNA and only one strand is copied.
• Why only one strand is copied:
1. Copying both strands would produce RNA molecules with different sequences (due to different DNA templates), potentially coding for two different proteins from one segment, complicating machinery.
2. Two complementary RNA molecules would form double-stranded RNA, preventing translation into protein.

12. Transcription Unit

• Defined by three regions in DNA:
1. A Promoter: Provides binding site for RNA polymerase. Located towards the 5'-end (upstream) of the structural gene (relative to the coding strand). Defines the template and coding strands.
2. The Structural gene: The segment of DNA being transcribed.
3. A Terminator: Usually defines the end of transcription. Located towards the 3'-end (downstream) of the coding strand.
• DNA Strands in a Transcription Unit:
• Template Strand: Polarity 3'→5' . Acts as the template for RNA synthesis. RNA polymerase polymerizes 5'→3'.
• Coding Strand: Polarity 5'→3' . Has the same sequence as the transcribed RNA (except T instead of U). Does not code for the RNA but is the reference point for defining the transcription unit (promoter/terminator positions).

13. Transcription Unit and the Gene

• Gene: The functional unit of inheritance. Located on DNA.
• DNA sequence coding for tRNA or rRNA also defines a gene.
• Cistron: A segment of DNA coding for a polypeptide.
• Structural Gene Types:
• Monocistronic: Codes for a single polypeptide (mostly in eukaryotes).
• Polycistronic: Codes for multiple polypeptides (mostly in bacteria/prokaryotes).
• Split Genes (Eukaryotes): Monocistronic genes have interrupted coding sequences.
• Exons: Coding sequences; appear in mature RNA.
• Introns: Intervening (non-coding) sequences; do not appear in mature RNA.
• Regulatory sequences (promoters, terminators) also affect inheritance. Sometimes loosely called "regulatory genes" though they don't code for RNA/protein.

14. Types of RNA and the Process of Transcription

• Types of RNA in Bacteria:
• mRNA (messenger RNA): Provides the template for protein synthesis.
• tRNA (transfer RNA): Brings amino acids and reads the genetic code.
• rRNA (ribosomal RNA): Plays structural and catalytic roles during translation.
• Transcription in Bacteria:
• Single DNA-dependent RNA polymerase catalyzes transcription of all RNA types.
• Initiation: RNA polymerase binds to the promoter. It associates transiently with an initiation-factor (σ). Uses nucleoside triphosphates as substrates. Polymerizes 5'→3'. Facilitates helix opening.
• Elongation: RNA polymerase moves along the template. Only a short RNA stretch remains bound to the enzyme.
• Termination: Polymerase reaches the terminator region. The nascent RNA falls off, and RNA polymerase detaches. It associates transiently with a termination-factor (ρ).
• Transcription and translation can be coupled in bacteria because there is no nucleus and mRNA does not require processing.
• Transcription in Eukaryotes: More complex.
• Multiple RNA polymerases in the nucleus:
• RNA polymerase I: Transcribes rRNAs (28S, 18S, 5.8S).
• RNA polymerase III: Transcribes tRNA, 5SrRNA, snRNAs.
• RNA polymerase II: Transcribes precursor of mRNA, called heterogeneous nuclear RNA (hnRNA) .
• Processing of Primary Transcript (hnRNA) :
• Primary transcript contains both exons and introns and is non-functional.
• Splicing: Introns are removed, and exons are joined in a defined order.
• Capping: Unusual nucleotide (methyl guanosine triphosphate) added to the 5'-end .
• Tailing: Adenylate residues (200-300) added to the 3'-end in a template-independent manner.
• Fully processed hnRNA is called mRNA and is transported out of the nucleus for translation.
• Significance of Eukaryotic Complexities: Split genes and splicing may represent ancient features and the dominance of the RNA-world.

15. Genetic Code

• Transfer of genetic information from nucleotide polymer (DNA/RNA) to amino acid polymer (protein).
• No direct complementarity between nucleotides and amino acids.
• Evidence: Change in nucleic acids linked to change in amino acids in proteins.
• Proposition: A genetic code directs amino acid sequence during protein synthesis.
• Deciphering the code was challenging, involving multiple disciplines.
• Triplets: George Gamow suggested the code must be a triplet (3 nucleotides) to code for 20 amino acids (4³ = 64 codons).

Experimental Work:

• Har Gobind Khorana: Chemical methods to synthesize RNA molecules with defined base combinations.
• Marshall Nirenberg: Cell-free system for protein synthesis to decipher codes.
• Severo Ochoa: Enzyme (polynucleotide phosphorylase) to polymerize RNA with defined sequences.

Salient Features of Genetic Code:

• Triplet: Code is a triplet of bases.
• 61 codons code for amino acids.
• 3 codons are stop codons (do not code for amino acids).
• Degenerate: Some amino acids are coded by more than one codon.
• Read in mRNA in a contiguous fashion (no punctuations).
• Nearly universal: Same codon codes for the same amino acid from bacteria to humans (e.g., UUU codes for Phenylalanine). Exceptions exist (mitochondrial codons, some protozoans).
• AUG has dual functions: Codes for Methionine (Met) and acts as initiator codon .
• UAA, UAG, UGA are stop/terminator codons .

16. Mutations and Genetic Code

• Relationship between genes and DNA understood via mutation studies.
• Point Mutations: Change of a single base pair.
• Example: Sickle cell anemia – single base change in beta globin gene changes glutamate to valine.
• Frameshift Mutations: Insertion or deletion of bases that changes the reading frame.
• Insertion or deletion of one or two bases changes the reading frame from that point onwards.
• Insertion or deletion of three (or multiples of three) bases inserts or deletes one or multiple codons, but the reading frame from that point onwards remains unaltered.

17. tRNA – the Adapter Molecule

• Francis Crick postulated an adapter molecule to read the genetic code and link it to specific amino acids.
• tRNA (transfer RNA, previously called sRNA) was identified as this adapter.
• Structure/Function:
• Has an anticodon loop with bases complementary to the genetic code (codon) in mRNA.
• Has an amino acid acceptor end to which it binds a specific amino acid.
• tRNAs are specific for each amino acid .
• There is a specific initiator tRNA for the start codon AUG.
• There are no tRNAs for stop codons .
• Secondary structure resembles a clover-leaf. Actual structure is compact, like an inverted L.

18. Translation

• Process of polymerization of amino acids to form a polypeptide .
• Amino acid sequence is dictated by the sequence of bases in the mRNA.
• Amino acids joined by peptide bonds . Requires energy.
• Charging of tRNA (Aminoacylation): Amino acids activated in presence of ATP and linked to their cognate tRNA.
• Ribosome: The cellular factory for protein synthesis.
• Composed of structural RNAs (rRNAs) and proteins (~80 different proteins).
• Exists as two subunits (large and small) in inactive state.
• Small subunit binds to mRNA to begin translation.
• Large subunit has sites for amino acids/tRNAs to bind close together for peptide bond formation.
• Ribosome acts as a catalyst: 23S rRNA in bacteria is a ribozyme catalyzing peptide bond formation.
• Translational Unit: Sequence of RNA flanked by a start codon (AUG) and a stop codon , coding for a polypeptide.
• Untranslated Regions (UTRs): Additional sequences in mRNA not translated. Present at both 5'-end (before start codon) and 3'-end (after stop codon). Required for efficient translation.
• Process:
• Initiation: Ribosome binds to mRNA at the start codon (AUG), recognized by initiator tRNA.
• Elongation: Complexes of amino acid-tRNA bind sequentially to appropriate codons on mRNA via complementary base pairing of anticodon. Ribosome moves along mRNA (codon to codon). Amino acids added one by one.
• Termination: A release factor binds to the stop codon. This terminates translation and releases the completed polypeptide from the ribosome.

19. Regulation of Gene Expression

• Regulation can occur at various levels.
• Eukaryotes: Regulation at multiple levels:
• Transcriptional level (primary transcript formation).
• Processing level (splicing regulation).
• Transport of mRNA from nucleus to cytoplasm.
• Translational level.
• Genes expressed based on function or metabolic/physiological/environmental conditions. Development/differentiation also involves coordinated gene regulation.
• Prokaryotes: Control of transcriptional initiation is the predominant site for regulation.
• Activity of RNA polymerase at a promoter regulated by accessory proteins (activators - positive, repressors - negative).
• Accessibility of promoter regions regulated by interaction of proteins with operators .
• Operator region is adjacent to promoter in most operons .
• Operator sequences bind repressor protein .
• Each operon has specific operator and repressor (e.g., lac operator binds lac repressor).
• Operon: Arrangement where a polycistronic structural gene is regulated by a common promoter and regulatory genes. Common in bacteria. Examples: lac, trp, ara, his, val operons.

20. The Lac Operon

• Elucidated by Francois Jacob and Jacque Monod. A transcriptionally regulated system.
• Involves lactose metabolism in E. coli .
• Components:
• Regulatory gene (i gene): Codes for the repressor protein. (i stands for inhibitor). Synthesized constitutively (all the time).
• Structural genes (z, y, a): Polycistronic. Code for enzymes needed for lactose metabolism.
• z gene: Codes for beta-galactosidase (β-gal) - hydrolyzes lactose into galactose and glucose.
• y gene: Codes for Permease - increases cell permeability to β-galactosides (like lactose).
• a gene: Codes for Transacetylase .
• Promoter: Common promoter for the structural genes. Binding site for RNA polymerase.
• Operator: Region adjacent to the promoter where the repressor binds.
• Inducer: Lactose (or allolactose). Regulates switching on/off of the operon. It is the substrate for β-galactosidase.
• Mechanism of Regulation:
• Absence of Inducer (Lactose): The repressor protein synthesized from the i gene binds to the operator region. This prevents RNA polymerase from transcribing the structural genes. Operon is off.
• Presence of Inducer (Lactose): Lactose (or allolactose) enters the cell (requires low level of permease expression). The inducer interacts with and inactivates the repressor . The inactivated repressor cannot bind the operator. This allows RNA polymerase access to the promoter, and transcription proceeds. Structural genes (z, y, a) are transcribed, and enzymes for lactose metabolism are produced. Operon is on.
• Regulation by repressor is called negative regulation .
• This can be viewed as enzyme synthesis regulation by its substrate.

21. Human Genome Project (HGP)

• Ambitious project launched in 1990 to sequence the complete DNA sequence of the human genome.
• Genetic make-up lies in DNA sequences; differences between individuals are in DNA sequences.
• Called a mega project due to its magnitude.
• Scale: Human genome ~3 x 10⁹ bp. Estimated cost initially $3/bp, total ~9 billion US dollars. Generated enormous data requiring high-speed computation (storage, retrieval, analysis).
• Closely associated with the development of Bioinformatics .

Goals of HGP:

• Identify all human genes (estimated 20,000-25,000).
• Determine the sequence of 3 billion bp.
• Store data in databases.
• Improve data analysis tools.
• Transfer related technologies to other sectors.
• Address Ethical, Legal, and Social Issues (ELSI) .
• Coordination: Coordinated by U.S. Dept. of Energy and NIH, with major contributions from Wellcome Trust (U.K.), Japan, France, Germany, China, etc.. Completed in 2003.
• Applications: Revolutionize diagnosis, treatment, and prevention of disorders. Understand human biology. Understand non-human organisms for applications in healthcare, agriculture, energy, environment. Many model organisms also sequenced (bacteria, yeast, C. elegans , Drosophila , plants).
• Methodologies: Two major approaches:
• Expressed Sequence Tags (ESTs): Focused on identifying genes expressed as RNA.
• Sequence Annotation: Blindly sequencing the whole genome (coding and non-coding) and then assigning functions to regions.
• Sequencing Process: Total DNA isolated, converted to random fragments of smaller sizes. Fragments cloned in hosts (bacteria, yeast) using vectors ( BACs - bacterial artificial chromosomes, YACs - yeast artificial chromosomes) for amplification. Fragments sequenced using automated DNA sequencers based on Frederick Sanger's method . Sequences assembled using computer programs based on overlapping regions. Sequences annotated and assigned to chromosomes. Chromosome 1 was the last to be completed (May 2006).
• Mapping: Genetic and physical maps generated using polymorphism of restriction sites and repetitive DNA ( microsatellites ).

22. Salient Features of Human Genome

• Contains 3164.7 million bp .
• Average gene size ~3000 bases; largest is dystrophin (2.4 million bases).
• Estimated total number of genes: 30,000 (lower than previous estimates).
• 99.9% of nucleotide bases are the same in all humans .
• Functions unknown for >50% of discovered genes.
• Less than 2% of the genome codes for proteins .
• Large portion made of repeated sequences .
• Repetitive sequences: DNA stretches repeated many times. Thought to have no direct coding function. Shed light on chromosome structure, dynamics, evolution.
• Chromosome 1 has most genes (2968), Y has fewest (231).
• Identified ~1.4 million locations with single-base differences: SNPs (Single Nucleotide Polymorphisms) . Useful for finding disease-associated genes and tracing history.

23. DNA Fingerprinting

• Technique to identify differences in DNA sequences between individuals.
• Based on the fact that 99.9% of human DNA is the same, but the differences make individuals unique .
• Comparing whole genomes is expensive and daunting.
• DNA fingerprinting is a quick way to compare sequences.
• Focuses on identifying differences in specific regions called repetitive DNA .
• Repetitive DNA: Small DNA stretches repeated many times.
• Separated from bulk DNA by density gradient centrifugation as satellite DNA peaks.
• Classified (micro-satellites, mini-satellites) based on composition, length, repeat units.
• Normally do not code for proteins.
• Show high degree of polymorphism . Form the basis of DNA fingerprinting.
• Polymorphism (variation at genetic level): Inheritable mutations present in a population at high frequency (>0.01).
• Higher in non-coding DNA as mutations there often have no immediate effect on reproductive ability. Accumulate over generations.
• Technique (initially developed by Alec Jeffreys) :
• Uses a satellite DNA sequence as a probe, showing high polymorphism: Variable Number of Tandem Repeats (VNTR) .
• VNTR is a mini-satellite. Small sequence arranged tandemly in varying copy numbers between chromosomes and individuals. Size varies from 0.1 to 20 kb.
• Steps:
1. Isolation of DNA.
2. Digestion of DNA by restriction endonucleases .
3. Separation of DNA fragments by electrophoresis .
4. Transferring (blotting) fragments to synthetic membranes (e.g., nitrocellulose, nylon) ( Southern blot hybridisation ).
5. Hybridization using a labeled VNTR probe.
6. Detection of hybridised fragments by autoradiography .
• Result: Autoradiogram shows many bands of differing sizes, creating a characteristic pattern for an individual's DNA. This pattern differs between individuals, except identical twins.
• Increased Sensitivity: Use of Polymerase Chain Reaction (PCR) allows DNA fingerprinting from a single cell.
• Applications:
• Forensic science (identification tool using DNA from crime scenes).
• Paternity testing.
• Determining population and genetic diversities.
• Evolutionary biology.

Frequently Asked Questions (FAQs)

1. What is the Central Dogma of Molecular Biology?
• It describes the flow of genetic information.
• Information generally flows from DNA to RNA to Protein.
• Some viruses show a reverse flow from RNA to DNA.
2. Why is DNA considered a better genetic material than RNA for storage?
• DNA is chemically less reactive due to the absence of a 2'-OH group in its sugar.
• It is structurally more stable, partly due to being double-stranded and having Thymine instead of Uracil.
• Its stability makes it more suitable for long-term storage of genetic information.
3. What is the basis of DNA fingerprinting?
• It relies on the principle of DNA polymorphism, variations in DNA sequences among individuals.
• Specifically, it uses differences in repetitive DNA sequences like VNTRs, which have varying copy numbers between individuals.
• These variations produce a unique banding pattern used for identification.