Molecular Basis of Inheritance

The DNA

This chapter focuses on the molecular basis of inheritance, covering key topics such as DNA packaging, genetic material, replication, transcription, genetic code, translation, gene expression regulation, the Lac Operon, the Human Genome Project, and DNA fingerprinting.

DNA: The Blueprint of Life

Introduction to DNA

• In the last chapter, we learned about how traits are inherited.
• Mendel discovered the inheritance patterns, but the material (Genetic material) responsible for these patterns, was unknown.
• DNA (deoxyribonucleic acid) was later discovered as the genetic material.
• DNA and RNA (ribonucleic acid) are nucleic acids made of nucleotides.

DNA and RNA:

• DNA: Genetic material in most organisms.
• RNA: Acts as a messenger, adapter, structural, and sometimes catalytic molecule.

DNA Structure:

• DNA is a long chain (polymer) of nucleotides (deoxyribonucleotides).
• Length of DNA: equals to number of nucleotides or base pairs (bp).
  • Example: Human DNA has 3.3 × 10⁹ bp.
  • E. coli: 4.6 million base pairs
  • Bacteriophage lambda: 48,502 base pairs

Polynucleotide Chain:

• Nucleotide 3 components:
  1. Nitrogenous bases: Purines (Adenine, Guanine), Pyrimidines (Cytosine, Thymine, Uracil).
     • RNA has uracil instead of thymine.
     • Base pairing: Adenine pairs with Thymine, Guanine pairs with Cytosine.
     • Erwin Chargaff’s rule: The amount of adenine (A) = thymine (T), and the amount of guanine (G) = cytosine (C), i.e. [A] + [G] = [T] + [C] or [A]+[G] / [T] + [C] = 1
     • This means A pairs with T and G pairs with C, maintaining a 1:1 ratio, i.e. A = T (2 hydrogen bonds), C = G (3 H bonds)
  2. Pentose Sugar: Ribose in RNA, Deoxyribose in DNA
  3. Phosphate Group
• Nucleotides link together to form a chain through phosphodiester bonds.
• Nucleotides chains have a 5’-end and a 3’-end, determined by the position of the phosphate group and sugar.

Discovery of DNA Structure:

• Friedrich Meischer discovered DNA and named it ‘Nuclein’ in 1869.
• James Watson and Francis Crick proposed the Double Helix model in 1953.

Double-Helix Structure Features:

• Two polynucleotide chains (strands) with sugar-phosphate backbone and bases inside.
• Anti-parallel chains (5′ to 3′ and 3′ to 5′), i.e. The two strands of DNA run in opposite directions: one strand goes from 5′ to 3′ and the other from 3′ to 5′.
• Base pairs linked by hydrogen bonds (A-T, G-C).
• Right-handed coiled chains with 10 bp per turn.
• Helix pitch: 3.4 nm, distance between bp: 0.34 nm.
• Stacked base pairs add stability.

Packaging of DNA Helix

DNA Length and Packaging:

• DNA in a mammalian (human) cell is about 2.2 meters long.
• The cell nucleus is very small (around 10–6 meters).
• Question: How is such a long DNA packed into a tiny nucleus?

DNA in Prokaryotes (e.g., E. coli):

• E. coli DNA length is 1.36 mm.
• DNA is held in a region called the nucleoid.
• DNA (negative charge) is held by proteins (positive charge).
• DNA is organized in large loops.

DNA in Eukaryotes:

• More complex organization.
• DNA is wrapped around proteins called histones.

Histones and Nucleosomes:

• Histones are positively charged due to lysine and arginine amino acids.
• Histones form a unit of eight molecules called a histone octamer.
• DNA wraps around the histone octamer, forming a nucleosome.
• Nucleosomes look like “beads-on-a-string” under a microscope.
• Each nucleosome has 200 base pairs of DNA.

Chromatin Structure:

• Nucleosomes make up chromatin, seen as thread-like structures in the nucleus.
• Chromatin can be loosely or densely packed.
  • Loosely packed chromatin: is Euchromatin (lightly stained, active in transcription).
  • Densely packed chromatin: is Heterochromatin (darkly stained, inactive).

Further Packaging:

• Chromatin fibers are further coiled and condensed to form chromosomes during cell division.
• Additional proteins called Non-histone Chromosomal (NHC) proteins help in higher-level packaging.

By understanding these concepts, we can see how DNA is efficiently packed into the tiny space of the cell nucleus, allowing it to function properly in both prokaryotic and eukaryotic cells.

The Search for Genetic Material

• The journey to discover DNA as the genetic material took a long time.
• Initially, it wasn’t clear what carried genetic information in cells.

Transforming Principle:

• 1928, Frederick Griffith: experimented with Streptococcus pneumoniae (bacteria causing pneumonia).
  • S Strain: Smooth colonies with a mucous coat, causes pneumonia in mice (virulent).
  • R Strain: Rough colonies without a mucous coat, does not cause pneumonia (non-virulent).
• Griffith found that heat-killed S strain mixed with live R strain killed mice, i.e. Heat-killed S strain + live R strain = mice died.
• This suggested a “transforming principle” from the heat-killed S strain made R strain virulent.
• This indicated the transfer of transforming material or genetic material.
• But the nature of this genetic material was not yet known.

Biochemical Characterization:

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) discovered:
  • Purified proteins, DNA, and RNA from heat-killed S cells.
  • Found DNA alone from S strain transformed R strain into virulent S strain.
  • Proteins and RNA did not cause this transformation.
  • Enzymes that digest proteins and RNA didn’t stop transformation, but DNase (which digests DNA) did.

DNA is the Genetic Material:

Hershey-Chase Experiment

• 1952, Alfred Hershey and Martha Chase:
• Worked with bacteriophages (viruses that infect bacteria).
• Goal: To find out if genetic material was DNA or protein.
  • They used radioactive phosphorus (in DNA) and sulfur (in proteins) to label viruses.
  • Only radioactive DNA entered bacteria, not proteins.
  • Conclusion: DNA is the genetic material.

Properties of Genetic Material (DNA vs. RNA):

• Criteria for Genetic Material
  1. Replication: Must replicate itself.
  2. Stability: Must be chemically and structurally stable.
  3. Mutation: Must allow slow changes (mutations) for evolution.
  4. Expression: Must express itself in form of traits or Mendelian Characters.
• Both DNA and RNA can replicate and mutate.

DNA vs. RNA

Feature	DNA	RNA
Stability	More stable chemically and structurally	Less stable, more reactive
	Thymine adds stability	2′-OH group makes RNA more reactive
Function	Relies on RNA to make proteins	Directly codes for protein synthesis
	Preferred for storing genetic information	Better for transmitting genetic information
Mutation Rate	Mutates slower	Mutates faster
		Thus RNA viruses evolve quickly
Presence in Cells	Found in the nucleus	Found in the nucleus and cytoplasm
Base Composition	Contains thymine	Contains uracil
Replication	Directs its own replication due to base pairing	Directs its own replication due to base pairing
Role in Evolution	Provides stability for long-term genetic storage	Facilitates quick adaptation and evolution
Example of Use	Genetic material in most organisms	Genetic material in some viruses; messenger and adapter in protein synthesis
Structural Stability	Two strands (double helix)	Single strand

Key Points

• Replication: Both DNA and RNA can replicate due to base pairing.
• Storage vs. Transmission: DNA’s stability makes it ideal for long-term genetic storage; RNA is more suited for quick transmission of genetic information.
• Chemical Reactivity: DNA is less chemically reactive; RNA is more reactive and catalytic.
• Mutation and Evolution: DNA mutates slower; RNA mutates faster, aiding quick evolution.
• Protein Synthesis: RNA can directly code for proteins; DNA depends on RNA for protein synthesis.

RNA World

Which Came First: DNA or RNA?

• RNA was the first genetic material.
• RNA played key roles in essential life processes like metabolism and protein synthesis.

Roles of RNA:

• Genetic Material: RNA carried genetic information in early life forms and still do for some viruses.
• Catalyst: RNA acted like enzymes to speed up biochemical reactions.

Why DNA Evolved from RNA:

• RNA was reactive and unstable because it acted as a catalyst.
• DNA evolved from RNA, gaining stability through chemical modifications.
• DNA is more stable due to its chemical structure and double-stranded form.
• DNA’s double-stranded structure and repair mechanisms provide additional stability.

Key Points:

• RNA was crucial in early life for both genetic information and catalysis.
• DNA evolved from RNA to provide a more stable and reliable form of genetic material.

Central Dogma

• Proposed by Francis Crick
  • Describes the flow of genetic information within a biological system, i.e. DNA → RNA → Protein.
• Steps in Central Dogma:
  1. DNA to RNA (Transcription):
     • The process where DNA is used as a template to make RNA.
     • Occurs in the nucleus in eukaryotes.
  2. RNA to Protein (Translation):
     • The process where RNA is used to build proteins.
     • Occurs in the ribosome in the cytoplasm.
• Key Points:
  • DNA: Contains the genetic blueprint of an organism.
  • mRNA (Messenger RNA): Carries the genetic information from DNA to the ribosome.
  • tRNA (Transfer RNA): Brings amino acids to the ribosome to build proteins.
  • rRNA (Ribosomal RNA): Forms the core of the ribosome’s structure and catalyzes protein synthesis.
• Exceptions:
  • In some viruses, genetic information flows from RNA → DNA, called Reverse Transcription.

Transcription

What is Transcription?

• Transcription is the process of copying genetic information from one strand of DNA into RNA.
• Only a segment of DNA and one strand is copied.
• Uses the principle of Base complementarity (A pairs with U in RNA, instead of T).

Why Only One Strand?

• If both strands were copied, they would create different RNA sequences, coding for different proteins.
• Two RNA molecules would be complementary bind together and form a double strand, making them non-functional and thus stop protein formation..
• Thus Only one strand is copied to avoid making two different proteins from one DNA segment.

Transcription Unit

Three Main Parts:

1. Promoter:
   • Located at the start (5′ end) of the gene.
   • Provides the binding site (starting point) for RNA polymerase.
2. Structural Gene:
   • The part of DNA that is actually transcribed into RNA.
3. Terminator:
   • Located at the end (3′ end) of the gene.
   • Signals the end of transcription.
   • Thus acts as endpoint for transcription.

DNA Strands:

• Template Strand: 3′ → 5′, used by RNA polymerase to make RNA.
• Coding Strand: 5′ → 3′, same sequence as RNA (except T is replaced by U in RNA).

Transcription in Action

RNA Sequence Example:

• Given DNA:
  • Template Strand: 3′-ATGCATGCATGCATGCATGCATGC-5′
  • Coding Strand: 5′-TACGTACGTACGTACGTACGTACG-3′
• Transcribed RNA would be: 5′-UACGUACGUACGUACGUACGUACG-3′

Genes and Transcription Units

Gene Definition:

• Functional unit of inheritance located on DNA.
• Includes sequences or can code for for tRNA and rRNA or polypeptides..

Types of Genes:

• Monocistronic: Mostly in eukaryotes, codes for one polypeptide or one gene per transcription unit.
• Polycistronic: Mostly in prokaryotes, codes for multiple polypeptides or multiple genes per transcription unit.

Gene Structure:

• Exons: Coding sequences that appear in mature RNA.
• Introns: Non-coding sequences removed during RNA processing and thus do not appear in mature RNA.

Regulatory Sequences:

• Affect inheritance and gene expression.
• Sometimes called regulatory genes, even though they don’t code for proteins or RNA.

Summary

• Transcription: Copying one DNA strand into RNA.
• Transcription Unit: Includes promoter, structural gene, and terminator.
• Genes: Functional units on DNA, can be monocistronic or polycistronic.
• Gene Structure: Includes exons and introns, regulated by promoter and other sequences.

Types of RNA and Transcription

Types of RNA:

• mRNA (Messenger RNA): Provides the template for protein synthesis.
• tRNA (Transfer RNA): Brings amino acids and reads the genetic code.
• rRNA (Ribosomal RNA): Plays a structural and catalytic role during protein synthesis.

Transcription Process in Bacteria:

1. Initiation:
   • RNA polymerase binds to the promoter region on DNA.
   • RNA polymerase starts transcription by opening the DNA helix.
2. Elongation:
   • RNA polymerase adds nucleotides to form RNA chain, based on the DNA template.
   • A short stretch of RNA remains attached to the enzyme during this process.
3. Termination:
   • When RNA polymerase reaches the terminator region, the new RNA and the enzyme (RNA polymerase) detach and new RNA strand is released.
   • This ends transcription.

RNA Polymerase Helpers:

• Initiation Factor (σ): Helps RNA polymerase start transcription.
• Termination Factor (ρ): Helps RNA polymerase stop transcription.

Coupled Transcription and Translation in Bacteria::

• Transcription and translation occur in the same compartment, allowing them to happen simultaneously.
• Translation can start even before transcription finishes.

Transcription in Eukaryotes

Complexities in Eukaryotes:

• Multiple RNA Polymerases:
  • RNA Polymerase I: Transcribes rRNA (28S, 18S, 5.8S).
  • RNA Polymerase III: Transcribes tRNA, 5S rRNA, and snRNAs.
  • RNA Polymerase II: Transcribes hnRNA (precursor to mRNA).
• Processing of hnRNA:
  1. Splicing: Removes introns (non-coding regions) and joins exons (coding regions).
  2. Capping: Adds a special nucleotide (methyl guanosine triphosphate) to the 5′ end of hnRNA.
  3. Tailing: Adds a long chain of adenylate residues (200-300) to the 3′ end of hnRNA.
• Final mRNA: After processing, hnRNA becomes mRNA and is transported out of the nucleus for translation.

Significance of Complexities:

• The split-gene arrangement and presence of introns reflect ancient genome features.
• Splicing and RNA processing indicate the importance of RNA-based processes in living systems.

Summary

• Three Types of RNA: mRNA, tRNA, rRNA, each with different roles in protein synthesis.
• Transcription Steps: Initiation, Elongation, Termination.
• RNA Polymerase: Needs initiation and termination factors in bacteria.
• Eukaryotic Transcription: More complex, involves multiple RNA polymerases and processing steps for hnRNA.

Genetic Code

Concept of Genetic Code:

• Genetic Code: Directs the sequence of amino acids during protein synthesis.
• Triplet Nature: Three nucleotides make one codon.

Background

• During replication and transcription, nucleic acids copy to form another nucleic acid.
• Translation involves converting genetic information from nucleotides to amino acids.
• There is no direct complementarity between nucleotides and amino acids.
• Changes in genetic material lead to changes in amino acids in proteins

Proposition of Genetic Code:

• Scientists from multiple disciplines worked on this.
• George Gamow’s Proposition: Suggested the genetic code is made up of three nucleotides (triplet code).
  • Reason: 4 bases (A, U, C, G) need to code for 20 amino acids.
  • Combination: 4³ (64 codons), more than needed for 20 amino acids.

Proof and Deciphering the Genetic Code:

• Har Gobind Khorana: Synthesized RNA with specific bases combinations.
• Marshall Nirenberg: Developed a cell-free system for protein synthesis.
• Severo Ochoa‘s enzyme (polynucleotide phosphorylase) helped polymerize RNA (create RNA) with defined sequences. .

Salient Features of Genetic Code

1. Triplet Code (nature):
   • 61 codons for amino acids.
   • 3 codons are stop codons.
2. Degeneracy:
   • Some amino acids are coded by more than one codon.
3. Continuous Reading:
   • Codons in mRNA are read without punctuation.
4. Universality:
   • Same codon codes for the same amino acid in almost all organisms (e.g., UUU codes for Phenylalanine in bacteria to humans).
   • Thus most codons are universal across species.
   • Exceptions exist in mitochondrial codons and some protozoans.
5. AUG Codon:
   • Codes for Methionine.
   • Acts as an initiator codon.
6. Stop Codons (3):
   • UAA, UAG, UGA signal the end of protein synthesis, thus act as stop codons.
7. Total: 61 codons for 20 amino acids.

Example Sequences

• mRNA Sequence: AUG UUU UUC UUC UUU UUU UUC
  • Amino Acid Sequence: Met-Phe-Phe-Phe-Phe-Phe-Phe. (Met =Methionine, Phe = Phenylalanine).

Correlating Properties of Genetic Code:

• The code is degenerate (more than one codon can code for the same amino acid).
• The code is read in a contiguous fashion (no spaces or punctuation).

Mutations and Genetic Code

Understanding Mutations

• Mutations are changes in the DNA sequence.
• Large deletions and rearrangements can lead to the loss or gain of genes, affecting their functions.

Point Mutations

• Point mutations involve changes in a single base pair in DNA.
• Example: A point mutation in the beta globin gene changes the amino acid glutamate to valine, causing sickle cell anemia.

Frameshift Mutations

• Inserting or deleting bases changes the reading frame of genetic code.
• Example with Sentence:
  • Original sentence: RAM HAS RED CAP
  • Inserting ‘B’: RAM HAS BRE DCA P
  • Inserting ‘BI’: RAM HAS BIR EDC AP
  • Inserting ‘BIG’: RAM HAS BIG RED CAP
  • Deleting ‘R’: RAM HAS EDC AP
  • Deleting ‘E’: RAM HAS DCA P
  • Deleting ‘D’: RAM HAS CAP
• Conclusion: Insertion or deletion of one or two bases changes/sifts the reading frame (frameshift mutations). Inserting or deleting three bases doesn’t change the reading frame.

tRNA – The Adapter Molecule

• Function: Reads the genetic code and links it to amino acids.
• Discovery: tRNA known before the genetic code was postulated.
• Structure:
  • Anticodon Loop: Bases complementary to the code or matches the genetic code.
  • Amino Acid Acceptor End: Binds to specific amino acids.
  • Specificity: Each tRNA is specific to one amino acid.
  • Initiator tRNA: Specific tRNA for initiation.
  • No tRNAs: exist for stop codons.
• Appearance (shape):
  • Secondary Structure: Clover-leaf shape.
  • Actual Structure: Compact, looks like an inverted L.

Summary

• Genetic Code: Triplet code (3 nucleotides) for amino acids.
• Mutations: Change in DNA sequence affects protein synthesis.
• tRNA: Adapter molecule crucial for translating genetic code into proteins.

Translation

• Definition: Process of joining amino acids to form a polypeptide (a chain of amino acids).
• Order of Amino Acids: Defined by the sequence of bases in mRNA.
• Bond Formation: Amino acids joined by peptide bonds.

Steps in Translation

1. Activation of Amino Acids:
   • Requires energy from ATP.
   • Amino acids linked to tRNA (charging of tRNA or aminoacylation).
2. Formation of Peptide Bonds:
   • Energetically favored if charged tRNAs are close.
   • Catalyzed by ribosome.

Role of Ribosome

• Role: Ribosomes are the cellular machines that make proteins.
• Structure: Made of structural RNAs and about 80 proteins.
  • Subunits: have two subunits: Large subunit and small subunit.
  • The small subunit binds to mRNA to start translation.
• Function: Synthesizes proteins by reading mRNA.
• Catalytic Role: 23S rRNA in bacteria acts as an enzyme (ribozyme).

Translation Process

Translational Unit in mRNA

• Translational Unit: Part of mRNA that includes the start codon (AUG) and stop codon, coding for a polypeptide.
• Untranslated Regions (UTR):
  • Parts of mRNA before the start codon and after the stop codon.
  • Present at both 5′-end and 3′-end of mRNA.
  • They help in efficient translation.

Phases of Translation

1. Initiation:
   • Ribosome binds to mRNA at start codon (AUG).
   • Start codon is recognized by initiator tRNA.
2. Elongation:
   • tRNA-amino acid complexes bind to codons in mRNA.
   • Ribosome moves along mRNA from codon to codon.
   • Amino acids added one by one.
3. Termination:
   • A release factor binds to stop codon.
   • Translation ends, and polypeptide is released from ribosome.

Summary

• Translation: Converts genetic information in mRNA into a protein.
• Ribosome: Central role in protein synthesis.
• Process: Initiation, elongation, and termination.

Regulation of Gene Expression

What is Gene Expression?

• Definition: Gene expression means producing a polypeptide (protein) from a gene.
• Regulation: Can occur at various levels to control how much polypeptide is made.

Levels of Regulation in Eukaryotes

1. Transcriptional Level:
   • Control at the formation of the primary RNA transcript.
2. Processing Level:
   • Regulation during splicing of RNA.
3. mRNA Transport:
   • Moving mRNA from the nucleus to the cytoplasm.
4. Translational Level:
   • Control during the synthesis of proteins from mRNA.

Why is Gene Expression Regulated?

• To ensure that cells only make proteins when needed.
• Example: E. coli bacteria make an enzyme, beta-galactosidase, to break down lactose into galactose and glucose for energy. If there’s no lactose, the bacteria don’t make this enzyme.
• Gene Regulation in Development:
  • Coordinated regulation of many genes for development and differentiation of an embryo into an adult.

Regulation in Prokaryotes

Control at Transcription Initiation

• RNA polymerase starts transcription at a promoter region.
• Regulatory Proteins can help or hinder this process:
  • Activators help RNA polymerase start transcription.
  • Repressors block RNA polymerase to stop/decrease transcription..

Operons and Operators

• Operon: Group of genes regulated together.
  • Promoter: Region where RNA polymerase binds to start transcription.
  • Operator: Sequence near the promoter that binds repressor proteins.
• Example: Lac operon in E. coli.
  • Lac Operator: Binds only to the lac repressor.
  • Function: Controls genes needed to break down lactose.

Summary

• Gene Expression: Controlled at multiple levels.
• Prokaryotes: Mainly regulated at transcription initiation.
• Operons: Specific to each set of genes, like the lac operon in E. coli.

The Lac Operon

What is the Lac Operon?

• Scientists: Discovered by Francois Jacob and Jacques Monod.
• Purpose: A system to regulate gene transcription in bacteria.
• Common in Bacteria: Other examples include trp operon, ara operon, his operon, val operon, etc.

Components of the Lac Operon

One regulatory gene (i) and three structural genes (z, y, a).

• Regulatory Gene (i gene):
  • Codes for the repressor protein.
• Structural Genes:
  • z gene: Codes for beta-galactosidase, which breaks down lactose into glucose and galactose.
  • y gene: Codes for permease, which helps lactose enter the cell.
  • a gene: Codes for transacetylase (helps in lactose metabolism).

How the Lac Operon Works

• When Lactose present (Lactose as Inducer):
  • Inducer: Lactose can turn the operon on.
  • Process:
    1. In the absence of glucose, lactose enters the bacterial cell.
    2. Lactose binds to the repressor protein, inactivating it.
    3. The inactivated repressor cannot bind to the operator region.
    4. RNA polymerase can now transcribe the structural genes.
    5. The cell produces enzymes to break down lactose.
• No Lactose present (Repressor Protein in action):
  1. Repressor protein being made continuously by the i gene.
  2. The repressor protein binds to the operator region.
  3. This blocks RNA polymerase from transcribing the genes.

Key Points

• Negative Regulation: The lac operon is primarily regulated by the repressor protein, thus an example of negative regulation..
• Function: The operon ensures enzymes needed for lactose metabolism are produced only when lactose is present.
• Inducers allowing gene expression: Only lactose and allolactose can inactivate the repressor, not glucose or galactose.

By understanding the lac operon, we learn how bacteria efficiently manage their resources and adapt to their environment by regulating gene expression based on the availability of nutrients.

Human Genome Project

What is the Human Genome Project (HGP)?

• Purpose: To find out the complete DNA sequence of the human genome.
• Start Year: 1990.
• Completion: 2003 (13-year project).

Why Sequence the Human Genome?

• The genetic make-up of an organism is in its DNA sequences.
• Differences between individuals are due to differences in their DNA sequences.

Goals of the HGP

1. Identify Genes: Find all the 20,000-25,000 genes in human DNA.
2. Sequence DNA: Determine the sequences of the 3 billion base pairs in human DNA.
3. Store Information: Store DNA sequence information in databases.
4. Data Analysis: Improve tools for analyzing this data.
5. Technology Transfer: Share related technologies with other industries.
6. Address Issues: Consider ethical, legal, and social issues related to the project.

Scale of the Project

• DNA Bases: Human genome has about 3 billion base pairs.
• Cost: Initially estimated at $3 per base pair, totaling about 9 billion US dollars.
• Data Storage: Would require 3300 books, each with 1000 pages, to store the DNA sequence from a single human cell.

International Collaboration

• Main Coordinators: U.S. Department of Energy and the National Institutes of Health.
• Major Partner: Wellcome Trust (U.K.).
• Other Contributors: Japan, France, Germany, China, and others.

Benefits of the HGP

• Medical Advances: Improved ways to diagnose, treat, and prevent various disorders.
• Understanding Biology: Insights into human biology and other organisms.
• Applications: Useful in healthcare, agriculture, energy production, and environmental solutions.

Methodologies

1. Expressed Sequence Tags (ESTs): Focus on identifying genes expressed as RNA.
2. Whole Genome Sequencing: Sequencing the entire genome, including coding and non-coding regions.

Sequencing Process

• DNA Fragmentation: Isolate & Breaking down DNA into smaller pieces for easier sequencing.
• Cloning: Amplifying DNA fragments using host like bacteria or yeast with BACs (bacterial artificial chromosomes) and YACs (yeast artificial chromosomes).
• Automated DNA Sequencers: Machines used for sequencing, based on a method developed by Frederick Sanger.
• Alignment and Annotation: Overlapping fragments are aligned using computer programs to assemble the full sequence and also assign (their positions) them to chromosomes.

Milestones

• Chromosome 1: The last human chromosome to be sequenced, completed in May 2006.
• Genetic and Physical Maps: Created using polymorphism in DNA sequences, important for DNA fingerprinting.

The Human Genome Project was a massive effort that has revolutionized our understanding of genetics and biology. Understanding our DNA sequence helps in diagnosing diseases, developing treatments, and much more.

Salient Features of the Human Genome

1. Size and Content:
   • The human genome has 3164.7 million base pairs (bp).
   • The average gene is about 3000 bases long, but this varies. The largest gene, dystrophin, has 2.4 million bases.
   • Total number of genes is around 30,000, much lower than previously thought (80,000 to 140,000).
2. Genetic Similarity:
   • Almost all (99.9%) nucleotide bases are the same in all humans.
3. Unknown Functions:
   • Over 50% of the discovered genes have unknown functions.
4. Protein-Coding Genes:
   • Less than 2% of the genome codes for proteins.
5. Repeated Sequences:
   • A large portion of the genome is made up of repeated sequences.
   • These repetitive sequences don’t code for proteins but are important for understanding chromosome structure, dynamics, and evolution.
6. Chromosomes:
   • Chromosome 1 has the most genes (2968), while the Y chromosome has the fewest (231).
7. Single Nucleotide Polymorphisms (SNPs):
   • About 1.4 million locations of single-base DNA differences, called SNPs, have been identified. SNPs help in finding disease-related genes and tracing human history.

Applications and Future Challenges

• Future Research: Understanding the DNA sequences will shape future biological research.
• New Approaches: Researchers can now study all genes in a genome or all transcripts in a tissue systematically instead of studying one or a few genes.
• Interconnected Networks: Study how tens of thousands of genes and proteins work together in networks to manage the chemistry of life.
• Interdisciplinary Collaboration: This will need collaboration from many scientists in both public and private sectors globally.

The Human Genome Project has provided a foundation for many scientific advancements, allowing researchers to approach biological questions in new, comprehensive ways.

DNA Fingerprinting

What is DNA Fingerprinting?

• Purpose: Quickly compares DNA sequences of individuals.
• DNA Uniqueness: 99.9% of human DNA is the same; differences in the remaining 0.1% make us unique.
• Focus: Looks at specific regions (sequences) called repetitive DNA, where small DNA stretches repeat many times in the genome, and are unique to each individual..

Satellite DNA

A type of repetitive DNA that consists of long tandem repeats and is typically found in centromeric and heterochromatic regions of chromosomes.

• Separation: Identified as different peaks during density gradient centrifugation.
• Types: Includes micro-satellites and mini-satellites.
• Role: Though they don’t code for proteins, they help in understanding chromosome structure and evolution.

Polymorphism

• Meaning: Variations in DNA sequences at a locus; arises due to mutations.
• Inheritance: Passed from parents to children, useful for identification in forensic science and paternity testing.
• Mutation Effects: More common in non-coding regions since these don’t affect reproductive ability.
• Types: Range from single nucleotide changes to large-scale changes.

Technique of DNA Fingerprinting

• Developer: Alec Jeffreys.
• Probe Used: Variable Number of Tandem (sequential) Repeats (VNTR).
• Steps Involved:
  1. Isolate DNA: Extract DNA from samples like blood, hair, or skin.
  2. Digest DNA: Cut the DNA into fragments using special enzymes (restriction enzymes).
  3. Separate DNA: Use electrophoresis to separate DNA fragments based on size.
  4. Transfer DNA: Blot (Transfer) the separated DNA onto a synthetic membrane.
  5. Hybridize DNA: Use a labeled probe that binds to specific DNA sequences.
  6. Detect DNA: Visualize the DNA fragments using autoradiography.

Characteristics of VNTR

• Meaning: A type of satellite DNA used in early DNA fingerprinting.
• Structure: Tandemly (sequential) repeated small DNA sequences.
• Polymorphism: High degree of variations; size varies from 0.1 to 20 kb.
• Pattern: Creates unique band patterns for individuals (except identical twins).

Applications

• Forensics: Identification in crime scenes.
  • Crime Scene Investigation: DNA from a crime scene is compared with suspects’ DNA. A match in the banding pattern identifies the suspect.
• Paternity Testing: Determine parentage by comparing DNA sequences.
• Genetic Mapping: Study genetic differences in populations.
• Genetics: Studying genetic diversity and population genetics.
• Sensitivity: Enhanced by PCR, allowing analysis from a single cell.

Important Terms

• VNTR (Variable Number of Tandem Repeats): A type of satellite DNA used in early DNA fingerprinting.
• Polymorphism: Genetic variations that occur in more than 1% of the population.
• PCR (Polymerase Chain Reaction): A technique to amplify small DNA samples, enhancing the sensitivity of DNA fingerprinting.

DNA fingerprinting has revolutionized the way we understand genetic differences, with wide applications in forensics and genetics, providing unique identification based on our DNA.

Chapter Summary:

• Nucleic acids are made up of nucleotides.
• DNA stores genetic information, while RNA helps in transfer and expression of information.
• DNA is chemically and structurally more stable compared to RNA.
• RNA evolved before DNA, and DNA was derived from RNA.
• DNA has a double-stranded helical structure held together by hydrogen bonds.
• Adenine pairs with Thymine, and Guanine pairs with Cytosine through hydrogen bonds.
• DNA replicates semiconservatively, guided by complementary base pairing.
• A segment of DNA that codes for RNA is called a gene.
• During transcription, DNA serves as a template for the synthesis of complementary RNA.
• In bacteria, transcribed mRNA is functional and can be directly translated.
• In eukaryotes, genes have coding sequences (exons) interrupted by non-coding sequences (introns).
• Introns are removed, and exons are joined to produce functional RNA through splicing.
• Messenger RNA contains base sequences that code for amino acids in triplets.
• Transfer RNA (tRNA) acts as an adapter molecule during translation.
• Ribosomes, with the help of mRNA, facilitate the joining of amino acids during protein synthesis.
• Ribosomal RNA (rRNA) acts as a catalyst for peptide bond formation.
• Transcription and translation are energetically expensive processes regulated primarily at the transcription level.
• Operons in bacteria are units of genes regulated together, like the Lac operon responsible for lactose metabolism.
• The amount of lactose in the environment regulates the Lac operon, controlling enzyme synthesis.
• The Human Genome Project aimed to sequence every base in the human genome.
• DNA Fingerprinting detects DNA sequence variations and has applications in forensic science, genetic diversity, and evolutionary biology.