Chapter 11: Molecular Basis of Inheritance

Introduction

Overview

• At Mendel's time, inheritance factors were unknown.
• DNA and RNA are nucleic acids made of nucleotides.
• DNA is genetic material in most organisms.
• RNA acts as genetic material in some viruses and functions mainly as a messenger, adapter, structural, or catalytic molecule.

Memory Aids

• DNA = Genetic blueprint in most life forms.
• RNA = Messenger and catalyst in cells.

Quick Checkpoints

• What are the two main nucleic acids?
• Which nucleic acid acts as genetic material in most organisms?

The DNA

Structure and Composition

• DNA is a long polymer of deoxyribonucleotides forming two complementary strands running antiparallel.
• Strands held by hydrogen bonds between opposite nitrogen bases.
• Four types of nucleotides form DNA.
• DNA length is measured by number of nucleotides or base pairs (bp).
• Examples: Φ x174 (5386 nucleotides), lambda phage (48502 bp), Escherichia coli (4.6 million bp), human haploid DNA (3.3 billion bp).

Memory Aids

• DNA strands run antiparallel and complement each other.
• Base pairs define DNA length and organism specificity.

Quick Checkpoints

• What is the orientation of DNA strands?
• How is DNA length defined?

Structure of Polynucleotide Chain

Components and Linkages

• Nucleotide = nitrogenous base + pentose sugar + phosphate group.
• Purines: adenine, guanine; Pyrimidines: cytosine, uracil (RNA), thymine (DNA).
• Cytosine common to DNA and RNA; uracil replaces thymine in RNA.
• Base linked to 1′C sugar via N-glycosidic linkage forming nucleoside.
• Phosphate linked to 5′-OH of nucleoside via phosphodiester bond forms nucleotide.
• Nucleotides linked 3′–5′ phosphodiester form dinucleotides and polynucleotide chains.
• RNA nucleotides have extra –OH at 2′ position; DNA identified by Friedrich Miescher in 1869 as 'Nuclein'.

Memory Aids

• Nucleotide = Base + Sugar + Phosphate.
• RNA has uracil; DNA has thymine.
• Phosphodiester bonds link nucleotides 3′ to 5′.

Quick Checkpoints

• What are the three parts of a nucleotide?
• Which base replaces thymine in RNA?
• What type of bond links nucleotides?

Structure of DNA

Double Helix Model

• Proposed by James Watson and Francis Crick in 1953, based on X-ray data by Wilkins and Franklin.
• Chargaff's rules: purines = pyrimidines (A + G = T + C), A = T, G = C.
• Base ratio A + T / G + C varies by species but constant within species.
• Each DNA strand acts as template for identical daughter DNA.
• Features:
• Two polynucleotide chains with sugar-phosphate backbone; bases inside.
• Chains are antiparallel (5′→3′ and 3′→5′).
• Bases paired by hydrogen bonds: A-T (2 bonds), G-C (3 bonds); purine pairs with pyrimidine.
• Right-handed double helix with 3.4 nm pitch and ~10 bp per turn.
• Base pairs stack for stability.

Memory Aids

• DNA strands are antiparallel and complementary.
• A pairs with T; G pairs with C.
• Double helix is right-handed with 10 bp per turn.

Quick Checkpoints

• Who proposed the DNA double helix model?
• What are Chargaff's base pairing rules?
• Describe the polarity of DNA strands.

Central Dogma

Genetic Information Flow

• Proposed by Francis Crick.
• Genetic information flows: DNA → RNA → Protein.
• Steps: DNA replication, transcription (DNA to mRNA), translation (mRNA to protein).
• In some viruses, reverse flow occurs: RNA → DNA (reverse transcription).

Memory Aids

• DNA → RNA → Protein is the central dogma.
• Reverse transcription occurs in some viruses.

Quick Checkpoints

• What is the direction of genetic information flow?
• What is reverse transcription?

Packaging of DNA Helix

DNA Length and Compaction

• DNA length = number of base pairs × distance between base pairs.
• Typical mammalian cell DNA length ≈ 2.2 meters.
• DNA is compacted to fit in small nucleus.

Memory Aids

• DNA is very long but tightly packed.

Quick Checkpoints

• How is DNA length calculated?
• Why is DNA packaging necessary?

DNA Packaging in Prokaryotes

Supercoiling and Nucleoid

• DNA is supercoiled with help of RNAs and non-histone proteins like polyamines.
• Compacted DNA mass is called nucleoid.

Memory Aids

• Prokaryotic DNA is supercoiled and forms nucleoid.

Quick Checkpoints

• What is nucleoid?
• How is prokaryotic DNA compacted?

DNA Packaging in Eukaryotes

Histones and Nucleosomes

• DNA wraps around histones (lysine and arginine rich proteins).
• Histones form octamer (eight molecules) called histone octamer.
• DNA + histone octamer = nucleosome.
• Five histone types: H₁, H₂A, H₂B, H₃, H₄; H₂A, H₂B, H₃, H₄ form octamer.
• Typical nucleosome contains ~200 bp DNA.
• Nucleosomes form chromatin, visible as thread-like bodies in nucleus.
• Chromatin appears as 'beads-on-string' under electron microscope.
• Chromatin fibers coil and condense into chromosomes during metaphase.
• Non-histone chromosomal proteins (NHC) assist higher-level packaging.
• Chromatin types: euchromatin (loosely packed, active), heterochromatin (densely packed, inactive).
• DNA between nucleosomes is linker DNA or inerbead DNA, bearing H₁ histone (plugging and marker protein).
• Nucleosome + linker DNA = chromatosome.

Memory Aids

• Histones package DNA into nucleosomes.
• Chromatin = nucleosomes + linker DNA.
• Euchromatin = active; heterochromatin = inactive.

Quick Checkpoints

• What proteins help package eukaryotic DNA?
• Define nucleosome and chromatosome.
• Difference between euchromatin and heterochromatin?

Transforming Principle

Key Findings

• 1928: Frederick Griffith studied Streptococcus pneumoniae with two strains: S-type (virulent, smooth colonies) and R-type (non-virulent, rough colonies).
• Heat-killed S strain alone did not kill mice; live R strain alone did not kill mice.
• Mix of heat-killed S strain and live R strain killed mice, showing transformation of R to virulent S type.
• Conclusion: A 'transforming principle' from dead S strain transferred genetic material to R strain, enabling virulence.

Memory Aids

• Griffith’s experiment: Dead S + Live R = Dead mice (transformation).
• Transforming principle = genetic material transfer.

Quick Checkpoints

• What are the two strains of Streptococcus pneumoniae?
• What did the heat-killed S strain demonstrate?
• What is the significance of the transforming principle?

Biochemical Characterisation of Transforming Principle (Avery Experiment)

Key Findings

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) purified proteins, DNA, and RNA from heat-killed S cells.
• Only DNA caused transformation of live R cells into S cells.
• Protease and RNase treatments did not stop transformation; DNase treatment did.
• Conclusion: DNA is the hereditary material, not protein or RNA.

Memory Aids

• DNase stops transformation → DNA is genetic material.
• Protease and RNase do not affect transformation.

Quick Checkpoints

• Which enzyme inhibited transformation?
• What was the role of protease and RNase?
• What did Avery’s experiment prove about genetic material?

DNA is the Genetic Material (Hershey Chase Experiment)

Key Findings

• Alfred Hershey and Martha Chase (1952) used bacteriophages to prove DNA is genetic material.
• Phages labeled with radioactive phosphorus (³²P) in DNA and radioactive sulfur (³⁵S) in protein.
• Only radioactive DNA entered Escherichia coli cells, not protein.
• Conclusion: DNA carries genetic information, not protein.

Memory Aids

• ³²P labels DNA; ³⁵S labels protein.
• DNA enters bacteria; protein does not.
• DNA = genetic material.

Quick Checkpoints

• What isotopes were used to label DNA and protein?
• Which component entered bacterial cells?
• What was the conclusion of the Hershey-Chase experiment?

Properties of Genetic Material (DNA versus RNA)

Key Properties

• Some viruses use RNA as genetic material (e.g., Tobacco Mosaic virus, QB bacteriophage).
• Genetic material must: replicate, be chemically and structurally stable, allow mutation, and express Mendelian characters.
• DNA and RNA can replicate due to base pairing; proteins cannot.
• DNA is chemically more stable than RNA due to absence of reactive 2'-OH group and presence of thymine instead of uracil.
• RNA is reactive, catalytic, and degrades easily.
• RNA mutates faster; RNA viruses evolve faster.
• RNA directly codes proteins; DNA depends on RNA for protein synthesis.
• DNA is preferred for genetic information storage; RNA for transmission.

Memory Aids

• DNA = stable, stores info; RNA = reactive, transmits info.
• Thymine in DNA adds stability; uracil in RNA less stable.
• RNA mutates faster → faster evolution.

Quick Checkpoints

• What are the criteria for genetic material?
• Why is DNA more stable than RNA?
• How do RNA and DNA differ in mutation rates and roles?

RNA World

RNA as First Genetic Material

• RNA was the first genetic material supporting essential life processes like metabolism, translation, and splicing.
• RNA acted both as genetic material and catalyst but was chemically reactive and unstable.
• DNA evolved from RNA with chemical changes making it more stable.
• DNA is double-stranded with complementary strands and repair mechanisms to resist changes.

Types of Non-Genetic RNA

• Based on size and function, three main non-genetic RNAs: mRNA (~3.5%), tRNA (~15%), rRNA (~80%).
• mRNA: Transcribed from genes, serves as template for protein synthesis by ribosomes.
• tRNA: Also called soluble, acceptor, or adapter RNA; has amino acid binding site opposite anticodon.
• rRNA: Structural component of ribosomes, synthesized in nucleolus.

Memory Aids

• RNA = First genetic material and catalyst.
• DNA = Stable, double-stranded, repair-capable.
• mRNA = Messenger, carries code.
• tRNA = Transfer, carries amino acids.
• rRNA = Ribosome, protein factory.

Quick Checkpoints

• What roles did RNA play in early life?
• How is DNA chemically more stable than RNA?
• What are the main types of non-genetic RNA and their functions?
• Where is rRNA synthesized?

Replication

Process and Mechanism

• DNA replication is the process of making an identical copy of double-stranded DNA using the original DNA as a template.
• Watson and Crick proposed semiconservative replication: each new DNA molecule has one parental and one newly synthesized strand.
• Replication involves unwinding the double helix and synthesizing complementary strands.

Memory Aids

• "Semi" means half old, half new strands in each DNA molecule.
• Complementary base pairing: A pairs with T, G pairs with C.

Quick Checkpoints

• What is semiconservative replication?
• Which strands serve as templates during replication?
• How does base pairing ensure accurate copying?

The Experimental Proof (Meselson and Stahl Experiment)

Experiment and Findings

• Meselson and Stahl (1958) proved DNA replicates semi-conservatively using Escherichia coli and nitrogen isotopes.
• DNA labeled with heavy nitrogen (¹⁵N) was grown in light nitrogen (¹⁴N) medium.
• After one generation, DNA density was intermediate, showing hybrid strands with one old and one new strand.
• Subsequent generations showed a mix of hybrid and light DNA, confirming semiconservative replication.
• Taylor et al. confirmed semi-conservative replication in chromosomes of Vicia faba using radioactive thymidine.

Memory Aids

• Heavy nitrogen (¹⁵N) labels old DNA strands.
• Hybrid DNA has one heavy and one light strand.
• Density gradient centrifugation separates DNA by weight.

Quick Checkpoints

• What isotope was used to label DNA in the experiment?
• How does DNA density change after replication in light nitrogen?
• What does the presence of hybrid DNA indicate?

The Machinery and the Enzymes

Enzymes and Process

• DNA replication is energy-intensive and requires enzymes called catalysts.
• Main enzyme: DNA-dependent DNA polymerase synthesizes new DNA strands using a DNA template.
• Replication speed in E. coli is about 2000 base pairs per second.
• Energy comes from breakdown of deoxyribonucleotide triphosphates.
• Helicase unwinds DNA at origin of replication (Ori site).
• Single-stranded binding proteins stabilize separated strands.
• Topoisomerases relieve supercoiling by nicking and resealing DNA strands.
• Replication fork is Y-shaped where DNA strands separate and replication occurs.
• RNA primer initiates new DNA strand synthesis; DNA polymerase adds nucleotides 5' to 3'.
• Leading strand replicates continuously (template 3' to 5').
• Lagging strand replicates discontinuously in Okazaki fragments (template 5' to 3').
• DNA ligase joins Okazaki fragments.
• Replication starts at specific origin sites, not randomly.
• In eukaryotes, replication occurs during S-phase of the cell cycle.

Memory Aids

• Helicase = Unwinds DNA.
• SSB proteins = Stabilize single strands.
• Topoisomerase = Relieves supercoiling.
• DNA polymerase = Synthesizes DNA 5' to 3'.
• RNA primer = Starts DNA synthesis.
• Ligase = Joins DNA fragments.
• Leading strand = Continuous synthesis.
• Lagging strand = Discontinuous synthesis.

Quick Checkpoints

• Which enzyme unwinds the DNA helix?
• What stabilizes single DNA strands during replication?
• Why is RNA primer needed?
• How does replication differ on leading and lagging strands?
• When does DNA replication occur in eukaryotic cells?

Transcription

Process and Mechanism

• Transcription is copying genetic information from one DNA strand into RNA.
• Complementarity governs transcription; adenine pairs with uracil (not thymine).
• Only one DNA strand and a segment are transcribed, unlike replication which duplicates entire DNA.
• Both DNA strands are not copied because:
• If both strands were templates, they would produce different RNA sequences, complicating protein synthesis.
• Simultaneous RNA molecules would be complementary, forming double-stranded RNA, blocking translation.
• A transcription unit has three DNA regions: promoter, structural gene, terminator.
• Structural gene can be monocistronic (eukaryotes) or polycistronic (prokaryotes).
• Coding strand sequence matches RNA except thymine is replaced by uracil.

Memory Aids

• Transcription = DNA to RNA copying.
• Only one strand used to avoid conflicting proteins.
• Promoter starts, terminator ends transcription.
• RNA matches coding strand except T → U.

Quick Checkpoints

• Why is only one DNA strand transcribed?
• What are the three regions of a transcription unit?
• How does RNA sequence relate to the coding strand?

Types of RNA and Process of Transcription

RNA Types and Transcription Steps

• Bacteria have three RNA types: mRNA (template), tRNA (amino acid carrier), rRNA (structural/catalytic).
• Transcription enzyme: DNA-dependent RNA polymerase holoenzyme = core enzyme + sigma factor.
• Sigma factor directs RNA polymerase to promoter, enhancing specificity.
• Transcription stages:
• Initiation: RNA polymerase binds promoter (-35 and -10 regions), DNA locally untwists.
• Elongation: Sigma factor leaves after 8-9 nucleotides; RNA polymerase synthesizes RNA 5'→3', unwinding DNA ahead.
• Termination: Rho factor binds RNA, catches polymerase at pause site, unwinds DNA-RNA hybrid, releasing polymerase.
• In bacteria, transcription and translation occur in cytoplasm; mRNA requires no processing.

Memory Aids

• 3 RNA types: mRNA (message), tRNA (transfer), rRNA (ribosome).
• Holoenzyme = core + sigma factor.
• Stages: Initiation (-35/-10), Elongation (5'→3'), Termination (rho factor).
• Bacterial mRNA is ready for translation immediately.

Quick Checkpoints

• What roles do the three RNA types play?
• What is the function of sigma factor?
• Describe the three transcription stages.
• Where does bacterial transcription occur?

Transcription in Eukaryotes

RNA Polymerases and mRNA Processing

• Three RNA polymerases in nucleus:
• RNA polymerase I: transcribes rRNAs (28S, 18S, 5.8S).
• RNA polymerase II: transcribes precursor mRNA (hnRNA).
• RNA polymerase III: transcribes tRNA, 5S rRNA, snRNA.
• Transcription occurs in nucleus; mRNA moves to cytoplasm for translation.
• mRNA maturation includes:
• 5' cap addition (7-methyl guanosine).
• 3' poly-A tail addition (200-300 adenines).
• Removal of introns by RNA splicing.
• Joining of exons by ligase to form mature mRNA.
• Primary transcript called heterogeneous nuclear RNA (hnRNA) or pre-mRNA.
• Splicing shows RNA world dominance.

Memory Aids

• RNA polymerase I = rRNA, II = pre-mRNA, III = tRNA + 5S rRNA + snRNA.
• mRNA processing: cap, tail, splicing.
• hnRNA = pre-mRNA with introns and exons.
• Splicing removes introns, joins exons.

Quick Checkpoints

• Which RNA polymerase transcribes pre-mRNA?
• What modifications occur during mRNA maturation?
• What is hnRNA?
• Why is RNA splicing important?

Genetic Code

Characteristics

• Genetic code is the codon representation decoding RNA into polypeptides.
• Code consists of three nucleotides (triplet code) producing 64 codons for 20 amino acids.
• Universal: Same codon codes same amino acid in all organisms (e.g., UUU codes phenylalanine).
• Degeneracy: Multiple codons can code for one amino acid.
• Non-overlapping: Each base is part of only one codon.
• Non-ambiguous: One codon codes for one amino acid, except AUG which can code methionine (start) or valine (internal).
• Initiator codon: Usually AUG, starts protein synthesis coding methionine.
• Non-sense codons: UAG, UAA, UGA signal termination of protein synthesis.
• Wobble hypothesis: Third base of codon binds loosely allowing one tRNA to recognize multiple codons differing at third base.
• First codon discovered: UUU; Nobel prize awarded to Holley, Nirenberg, Khorana in 1968.

Memory Aids

• Triplet code = 3 bases per codon.
• Universal code = same in all organisms.
• Degeneracy = multiple codons for one amino acid.
• Wobble = flexibility at third base.
• Start codon = AUG (Methionine).
• Stop codons = UAG, UAA, UGA.

Quick Checkpoints

• What is the length of a codon?
• Which codon initiates protein synthesis?
• Define degeneracy of genetic code.
• What are non-sense codons?
• Explain wobble hypothesis briefly.

Mutations and Genetic Code

Types and Effects

• Point mutation: Single base change causing amino acid substitution (e.g., glutamate to valine causes sickle cell anemia).
• Frameshift mutation: Insertion/deletion of 1 or 2 bases shifts reading frame, altering protein.
• Insertion/deletion of multiples of 3 bases does not shift frame, proving codon is triplet and read continuously.
• Non-sense mutation: Mutation creates stop codon, halting protein synthesis (e.g., UUA to UAA).
• Mis-sense mutation: Codon change leads to different amino acid (e.g., UUA Leucine to UUU Phenylalanine).
• Same sense mutation: Silent mutation; codon changes but amino acid remains same due to degeneracy (e.g., UUA to UUG).

Memory Aids

• Point mutation = single base change.
• Frameshift = insertion/deletion not multiple of 3.
• Non-sense = stop codon formed.
• Mis-sense = amino acid changes.
• Silent mutation = no amino acid change.

Quick Checkpoints

• What causes sickle cell anemia at genetic level?
• Define frameshift mutation.
• Difference between non-sense and mis-sense mutation?
• What is silent mutation?

tRNA – the Adapter Molecule

Structure and Function

• tRNA reads genetic code and binds specific amino acids; also called soluble RNA (sRNA).
• Secondary structure resembles clover-leaf; actual shape is compact inverted L.
• Has anticodon loop with bases complementary to mRNA codon.
• Has amino acid acceptor end for binding amino acids.
• Each tRNA is specific for one amino acid.
• Initiator tRNA starts translation; no tRNA for stop codons.

Memory Aids

• tRNA = adapter between mRNA codon and amino acid.
• Anticodon pairs with mRNA codon.
• Initiator tRNA starts protein synthesis.
• No tRNA for stop codons.

Quick Checkpoints

• What is the role of tRNA in protein synthesis?
• Describe the structure of tRNA.
• What is an anticodon?
• Which tRNA initiates translation?
• Are there tRNAs for stop codons?

Translation

Process Overview

• Translation is the polymerisation of amino acids into a polypeptide.
• Amino acid sequence is determined by mRNA base sequence.
• Amino acids are joined by peptide bonds, requiring energy.
• Amino acids are activated with ATP and linked to their specific tRNA (charging/aminoacylation).
• Ribosome, made of rRNAs and ~80 proteins, synthesises proteins.
• Ribosome has two subunits: large and small; translation starts when small subunit binds mRNA.
• Large subunit has two sites for amino acid binding; ribosome acts as catalyst (ribozyme) for peptide bond formation.
• mRNA has untranslated regions (UTRs) at 5' and 3' ends for efficient translation.
• Translation starts at AUG codon recognised by initiator tRNA.
• Elongation: aminoacyl-tRNAs bind codons on mRNA; ribosome moves codon to codon adding amino acids.
• Termination: release factor binds stop codon, releasing polypeptide.

Memory Aids

• "Charging tRNA" = amino acid + tRNA + ATP.
• Ribosome = protein factory with two subunits.
• Start codon AUG = initiator tRNA binding site.
• Peptide bond formation catalysed by ribosomal RNA (ribozyme).
• UTRs ensure translation efficiency.

Quick Checkpoints

• What defines amino acid sequence in translation?
• What is the role of tRNA charging?
• How does ribosome catalyse peptide bond formation?
• What are untranslated regions in mRNA?
• How does translation terminate?

Protein Biosynthesis

Post-Translational Modifications

• Proteins made by ribosomes undergo modifications to become mature.
• Modifications are enzyme-catalysed and target specific protein sequences.

Memory Aids

• PTMs = protein maturation steps after synthesis.
• Enzymes recognise specific sequences for modification.

Quick Checkpoints

• What happens to proteins after synthesis?
• What catalyses post-translational modifications?

Polyribosomes

Simultaneous Translation

• Multiple ribosomes translate a single mRNA simultaneously.
• First ribosome moves past start codon, allowing next ribosome to bind.
• Cluster of ribosomes on mRNA is called polyribosome.
• Found in both prokaryotes and eukaryotes.

Memory Aids

• Polyribosome = many ribosomes on one mRNA.
• Increases protein production speed.

Quick Checkpoints

• What is a polyribosome?
• How does polyribosome formation increase protein synthesis?

Regulation of Gene Expression

Gene Expression and Regulation

• Genes express to perform specific functions, e.g., beta-galactosidase in E. coli hydrolyses lactose into glucose and galactose.
• Gene expression depends on metabolic, physiological, or environmental conditions.
• Two types of gene regulation: positive (inducible) and negative (repressible).
• Negative regulation: genes express until suppressed by a regulatory product.
• Positive regulation: genes remain off until induced.
• Eukaryotic regulation occurs at transcription, RNA processing, mRNA transport, and translation levels.
• In prokaryotes, gene expression is mainly controlled at transcription initiation.
• RNA polymerase activity at promoters is regulated by proteins binding to operators adjacent to promoters.
• Each operon has a specific operator and repressor, e.g., lac operator binds only lac repressor.

Memory Aids

• Negative = Repressible = Always ON until turned OFF.
• Positive = Inducible = Always OFF until turned ON.
• Prokaryotes control gene expression mainly at transcription initiation.
• Operators are specific to their repressors.

Quick Checkpoints

• What triggers gene expression in positive regulation?
• Where does eukaryotic gene regulation occur?
• What is the role of operator in prokaryotic gene regulation?
• How does negative regulation differ from positive regulation?

The Lac Operon

Structure and Function

• Discovered by Francois Jacob and Jacques Monod.
• Operon: cluster of genes under a single promoter, common in bacteria.
• Lac operon has one regulatory gene (i) and three structural genes (z, y, a).
• i gene codes for lac repressor protein.
• z gene codes for beta-galactosidase, hydrolyzing lactose into glucose and galactose.
• y gene codes for permease, increasing cell permeability to lactose.
• a gene codes for transacetylase, involved in lactose metabolism.
• Lactose acts as an inducer by binding to the repressor and inactivating it.
• In absence of glucose, lactose enters cell via permease and induces operon expression.
• Repressor binds operator to block RNA polymerase and prevent transcription.
• Inducer binding inactivates repressor, allowing transcription of z, y, a genes.
• Regulation by repressor is negative regulation.

Memory Aids

• i gene = inhibitor (repressor).
• z = zero (beta-galactosidase breaks lactose).
• y = yes (permease helps lactose entry).
• a = acetylase (transacetylase enzyme).
• Lactose = inducer that inactivates repressor.
• Negative regulation = repressor blocks transcription until inducer present.

Quick Checkpoints

• What genes are present in lac operon and their functions?
• How does lactose regulate lac operon?
• What is the role of the repressor protein?
• Define negative regulation in lac operon context.

Human Genome Project

Overview

• Launched in 1990 as a mega project called International Human Genome Sequencing Consortium.
• Aimed to identify all human genes, determine their functions, and understand organism formation.
• Coordinated by U.S. Department of Energy and National Institute of Health; completed in 2003.
• Closely linked to bioinformatics development.

Goals

• Identify 20,000-25,000 human genes.
• Sequence 3 billion DNA base pairs.
• Store data in databases.
• Improve data analysis tools.
• Transfer technology to industries.
• Address ethical, legal, social issues (ELSI).
• Develop genetic linkage map using genetic markers.
• Obtain physical map by cloning DNA into BACs and YACs.

Enabling Factors

• Genetic engineering for DNA isolation and cloning.
• Fast DNA sequencing techniques.
• Sequencing of model organisms like bacteria, yeast, Caenorhabditis elegans, Drosophila, rice, and Arabidopsis.

Memory Aids

• HGP = Human genes + sequencing + bioinformatics + ethics.
• Goals: Identify, Sequence, Store, Analyze, Transfer, Ethics, Maps.

Quick Checkpoints

• What is the main aim of HGP?
• Which organizations coordinated HGP?
• What are BACs and YACs used for?

Methodologies of HGP

Techniques Used

• Expressed Sequence Tags (ESTs) to identify genes expressed as RNA.
• Sequence Annotation to assign functions to coding and non-coding regions.
• Automated DNA sequencing based on Sanger's method.
• Generation of overlapping DNA fragments for sequencing.
• Computer programs for sequence alignment and annotation.
• Chromosome 1 sequencing completed in 2006 (last human chromosome sequenced).

Memory Aids

• ESTs focus on expressed genes.
• Sanger method for sequencing.
• Computers essential for sequence assembly.

Quick Checkpoints

• What is the role of ESTs?
• Why are computers needed in HGP?
• When was chromosome 1 sequencing completed?

Salient Features of HGP

Key Observations

• Human genome has 3,164.7 million nucleotide base pairs.
• Average gene size ~3000 bases; largest gene dystrophin (2.4 million bases).
• Estimated 30,000 genes; 99.9% nucleotide bases identical in all humans.
• Functions of over 50% genes unknown.
• Less than 2% genome codes for proteins.
• Repeated DNA sequences form large genome portion.
• Chromosome 1 has most genes (2968); Y chromosome has fewest (231).
• About 1.4 million single nucleotide polymorphisms (SNPs) identified.

Memory Aids

• Genome size ~3.2 billion bases.
• Most genes on chromosome 1, least on Y.
• SNPs = single base differences in DNA.

Quick Checkpoints

• How many base pairs are in the human genome?
• What percentage of genome codes for proteins?
• What are SNPs?

Applications of HGP

Uses

• Enables systematic, large-scale biological research.
• Allows study of all genes or transcripts in tissues/organs/tumors.
• Helps understand gene and protein networks in life chemistry.

Memory Aids

• HGP = Research scale-up + gene study + network understanding.

Quick Checkpoints

• How does HGP change biological research?
• What can be studied using HGP data?
• Why is understanding gene networks important?

DNA Fingerprinting

Concept and Process

• DNA fingerprinting identifies differences in repetitive DNA regions called Variable Number of Tandem Repeats (VNTRs).
• VNTR size ranges from 0.1 to 20 kb; repetitive DNA forms satellite DNA peaks separate from bulk genomic DNA.
• These sequences show high polymorphism, unique to individuals except identical twins.
• DNA from any tissue (blood, hair, skin, saliva, sperm) shows the same polymorphism pattern.
• Useful in forensic identification and genetic diversity studies.
• Polymorphism arises from mutations; multiple alleles exist at a locus with frequency >0.01.
• Developed by Alec Jeffreys using satellite DNA probes and Southern blot hybridization.
• Steps: DNA extraction, PCR amplification, restriction enzyme digestion, electrophoresis, denaturation, transfer to membrane (Southern blotting), probe hybridization, autoradiography.

Memory Aids

• VNTR = Variable Number of Tandem Repeats, key to uniqueness.
• Polymorphism = multiple alleles due to mutations.
• Southern blotting = transfer DNA to membrane for probe detection.
• Alec Jeffreys = pioneer of DNA fingerprinting.

Quick Checkpoints

• What is VNTR and its role in DNA fingerprinting?
• How does polymorphism contribute to individual identification?
• What are the main steps in DNA fingerprinting?
• Who developed DNA fingerprinting and what probe was used?

Southern Blotting

Technique and Steps

• Transfers DNA fragments from gel to membrane, preserving banding pattern.
• DNA immobilized on membrane for hybridization with labeled probes.
• Named after Edwin Southern (1975).
• Steps: DNA digestion by restriction enzymes, gel electrophoresis, transfer to nylon membrane, prehybridization with buffer, hybridization with specific probe, detection by X-ray or other methods.
• Used to detect specific DNA sequences within a mixture.

Memory Aids

• Southern blot = DNA transfer and detection.
• Restriction enzymes cut DNA into fragments.
• Probe binds only to complementary DNA sequences.
• Detection shows dark bands where probe hybridizes.

Quick Checkpoints

• What is the purpose of Southern blotting?
• How are DNA fragments separated before transfer?
• What role does the probe play in Southern blotting?
• How is the hybridized DNA detected?

DNA Sequencing by Sanger's Method

Process and Significance

• Determines exact nucleotide order (A, T, C, G) in DNA.
• Developed by F. Sanger in 1977 using enzymatic chain termination.
• Steps: DNA isolation, fragmentation, PCR amplification, denaturation, primer binding, DNA polymerase elongation with normal nucleotides and chain-terminating ddNTPs.
• ddNTPs lack 3' OH group, causing chain termination at random points.
• Fragments separated by gel electrophoresis; sequence deduced from fragment sizes.
• Essential for genome analysis and understanding biological processes.

Memory Aids

• ddNTPs stop DNA chain growth, enabling sequence determination.
• Chain termination method = Sanger sequencing.
• Gel electrophoresis separates fragments by size.
• Primer initiates DNA synthesis.

Quick Checkpoints

• What causes chain termination in Sanger sequencing?
• Why is gel electrophoresis used in sequencing?
• What is the role of primers in DNA sequencing?
• How does Sanger sequencing help in genome analysis?