DNA
Deoxyribonucleic acid (DNA) is the hereditary material in most organisms. It carries genetic instructions for growth, development, and reproduction. DNA is found in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells.
Structurally, DNA is a double-helix molecule composed of two long strands of nucleotides. Each nucleotide consists of three components: a nitrogenous base, a deoxyribose sugar, and a phosphate group. There are four nitrogenous bases—adenine (A), thymine (T), cytosine (C), and guanine (G). A always pairs with T, and C pairs with G, forming complementary base pairs held together by hydrogen bonds.
DNA replicates through a semi-conservative process where each strand serves as a template for a new complementary strand. This ensures genetic information is passed on accurately during cell division.
The function of DNA is to store genetic information and guide protein synthesis. The sequence of bases in DNA determines the sequence of amino acids in proteins. Genes, which are segments of DNA, code for specific proteins. The process of gene expression involves transcription (formation of mRNA from DNA) and translation (conversion of mRNA into proteins).
DNA is a stable molecule due to its double-stranded structure and hydrogen bonding. It can undergo mutations, which may lead to genetic variations. These variations drive evolution and influence hereditary traits.
In some viruses, RNA serves as the genetic material instead of DNA. However, in most living organisms, DNA is the primary carrier of genetic information.
Structure of Polynucleotide Chain
A polynucleotide chain is a long chain of nucleotides linked by phosphodiester bonds. DNA and RNA are both composed of polynucleotide chains, but they differ in structure and function.
Nucleotide Composition
Each nucleotide consists of three components:
- Nitrogenous Base: There are two types of nitrogenous bases:
- Purines (double-ring structure): Adenine (A) and Guanine (G)
- Pyrimidines (single-ring structure): Cytosine (C), Thymine (T) in DNA, and Uracil (U) in RNA
- Pentose Sugar: DNA contains deoxyribose, while RNA contains ribose. The absence of an oxygen atom in deoxyribose makes DNA more stable.
- Phosphate Group: The phosphate group provides the acidic nature to nucleic acids and forms the backbone of the polynucleotide chain.
Formation of Polynucleotide Chain
Nucleotides are linked by phosphodiester bonds, which form between the 3′ carbon of one sugar and the 5′ carbon of the next sugar. This linkage results in a sugar-phosphate backbone, providing stability to the chain.
The polynucleotide chain has two distinct ends:
- 5′ end: Has a free phosphate group attached to the 5′ carbon of the sugar.
- 3′ end: Has a free hydroxyl (-OH) group attached to the 3′ carbon of the sugar.
DNA is a double-stranded molecule with two complementary polynucleotide chains running in an antiparallel direction (one strand runs 5′ to 3′, the other 3′ to 5′). The two strands are held together by hydrogen bonds between complementary base pairs:
- Adenine (A) pairs with Thymine (T) via two hydrogen bonds.
- Cytosine (C) pairs with Guanine (G) via three hydrogen bonds.
The helical structure of DNA was proposed by Watson and Crick based on X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins.
Differences Between DNA and RNA Polynucleotide Chains
| Feature | DNA | RNA |
|---|---|---|
| Sugar | Deoxyribose | Ribose |
| Bases | A, T, C, G | A, U, C, G |
| Strands | Double-stranded | Single-stranded |
| Stability | More stable | Less stable |
| Function | Genetic material | Involved in protein synthesis |
Biological Significance of Polynucleotide Chains
Polynucleotide chains store genetic information and direct the synthesis of proteins. DNA’s double-stranded nature allows it to be replicated and transcribed into RNA. RNA then helps in translating genetic information into functional proteins.
Central Dogma of Molecular Biology
The central dogma of molecular biology explains the flow of genetic information in cells. It was proposed by Francis Crick and states that genetic information follows a unidirectional pathway:
DNA → RNA → Protein
- Replication: DNA makes an identical copy of itself.
- Transcription: DNA is transcribed into messenger RNA (mRNA).
- Translation: mRNA is translated into proteins at ribosomes.
Some viruses, like retroviruses, can reverse this flow using reverse transcription (RNA → DNA). However, the general principle remains that DNA holds genetic instructions, RNA carries them, and proteins execute cellular functions.
Packaging of DNA Helix
DNA is an extremely long molecule that needs to be efficiently packed within the nucleus of eukaryotic cells. This is achieved through a hierarchical system of compaction involving histones and chromatin fibers.
Levels of DNA Packaging
- Nucleosome Formation:
- DNA wraps around histone proteins to form nucleosomes.
- Each nucleosome consists of 8 histone proteins (2 copies of H2A, H2B, H3, and H4).
- Around 146 base pairs of DNA wrap around a nucleosome, forming a “beads-on-a-string” structure.
- Chromatin Fiber Formation:
- Histone H1 binds nucleosomes together, forming a 30 nm chromatin fiber.
- Looped Domains:
- The chromatin fiber forms loops attached to a protein scaffold.
- These loops further condense to form a 300 nm fiber.
- Chromosome Formation:
- During cell division, the chromatin condenses further into chromosomes.
- Fully condensed metaphase chromosomes have a 1400 nm thickness.
Prokaryotic vs. Eukaryotic DNA Packaging
- Prokaryotes have circular DNA, which is supercoiled with the help of proteins.
- Eukaryotes have linear DNA, tightly packed with histones into chromosomes.
DNA packaging is crucial for gene regulation. Loosely packed chromatin (euchromatin) is transcriptionally active, while tightly packed chromatin (heterochromatin) is inactive.
The Search for Genetic Material
The identification of DNA as the genetic material involved several key experiments:
- Griffith’s Experiment (1928):
- Frederick Griffith worked with Streptococcus pneumoniae and discovered transformation—a non-virulent strain became virulent when mixed with heat-killed virulent bacteria.
- Avery, MacLeod, and McCarty (1944):
- They identified DNA as the transforming principle by using enzymes to degrade different biomolecules.
- Hershey and Chase Experiment (1952):
- Used bacteriophages labeled with radioactive sulfur (for proteins) and phosphorus (for DNA).
- Only radioactive DNA entered bacterial cells, confirming that DNA is the genetic material.
These experiments established DNA as the carrier of genetic information, disproving the earlier belief that proteins were the genetic material.
Observations:
- When treated with protease (protein-degrading enzyme) → Mice died → Proteins were ruled out as the transforming principle.
- When treated with RNase (RNA-degrading enzyme) → Mice died → RNA was ruled out.
- When treated with DNase (DNA-degrading enzyme) → Mice survived → Transformation did not occur.
- Thus, DNA degradation prevented transformation, indicating that DNA is the genetic material responsible for transformation.
Biochemical Characterization:
To confirm their findings, Avery, MacLeod, and McCarty performed a biochemical analysis of the transforming substance:
- The transforming substance had the same chemical composition as DNA:
- Nitrogen to phosphorus ratio consistent with DNA.
- It gave a positive reaction to the Dische diphenylamine test, which is specific for DNA.
- The material was susceptible to DNase but not affected by proteases or RNase, confirming that it was DNA.
Conclusion:
The experiment led to the conclusion that:
- DNA, not proteins or RNA, is the transforming principle.
- DNA is the carrier of hereditary information, overturning the earlier belief that proteins were the genetic material.
- This experiment laid the foundation for future research, eventually confirmed by Hershey and Chase’s experiment in 1952.
The Genetic Material is DNA (Hershey and Chase Experiment)
Background:
The definitive proof that DNA is the genetic material came from the Hershey and Chase experiment in 1952. Their work involved studying the life cycle of bacteriophages (viruses that infect bacteria).
Experimental Setup:
- They used T2 bacteriophage, which infects Escherichia coli (E. coli) bacteria.
- Bacteriophages consist of:
- A protein coat (capsid)
- Genetic material (DNA) inside the coat
Labeling the Phage Components:
To differentiate between the two biomolecules, they used radioactive labeling:
- Protein Labeling:
- They grew bacteriophages in a medium containing radioactive sulfur (³⁵S), which labels the protein coat since proteins contain sulfur.
- DNA Labeling:
- Another batch was grown in a medium containing radioactive phosphorus (³²P), which labels DNA, as phosphorus is present in nucleic acids but not in proteins.
Infection of Bacteria:
- The radioactive bacteriophages were allowed to infect E. coli cells.
- Following infection:
- The phages attached to the bacterial surface and injected their genetic material into the bacterial cell.
- The empty protein coats remained outside the bacterial cells.
Blending and Centrifugation:
- After infection, the mixture was agitated in a blender to detach the empty protein coats from the bacterial surface.
- The mixture was centrifuged:
- The heavier bacterial cells formed a pellet at the bottom.
- The lighter phage protein coats remained in the supernatant (liquid).
Observations:
- In the batch with ³⁵S-labeled proteins:
- Radioactivity was detected in the supernatant, indicating that the proteins did not enter the bacterial cells.
- In the batch with ³²P-labeled DNA:
- Radioactivity was found in the pellet, indicating that DNA had entered the bacterial cells.
- The infected bacteria produced new phages containing radioactive DNA, confirming that DNA is the genetic material.
Conclusion:
- DNA, not protein, is the genetic material responsible for carrying genetic information.
- When bacteriophages infect bacteria, only their DNA enters the host cell, while the protein coat remains outside.
- This experiment conclusively proved that DNA is the molecule of inheritance, providing further evidence that supported Avery, MacLeod, and McCarty’s earlier findings.
Properties of Genetic Material
A genetic material must fulfill the following criteria:
- Replication – It should be able to produce an identical copy of itself.
- Storage of Information – It should store genetic information for an organism’s growth and function.
- Expression of Information – It should express the stored information in the form of proteins.
- Mutation and Variation – It should be capable of undergoing mutations to allow evolution.
DNA vs RNA (Comparison in Tabular Form)
| Feature | DNA | RNA |
|---|---|---|
| Sugar | Deoxyribose | Ribose |
| Bases | A, T, C, G | A, U, C, G |
| Strands | Double-stranded | Single-stranded |
| Stability | More stable | Less stable |
| Genetic Material | Primary genetic material | Genetic material in some viruses (e.g., HIV) |
| Function | Stores genetic information | Helps in protein synthesis |
| Replication | Can self-replicate | Cannot self-replicate, synthesized from DNA |
Which is a Better Genetic Material: DNA or RNA?
- DNA is considered a better genetic material because:
- It is more stable due to the absence of an -OH group at the 2′ carbon.
- It has thymine instead of uracil, reducing mutation chances.
- It is double-stranded, allowing error correction and protection.
However, RNA was the first genetic material as it could both store genetic information and catalyze reactions.
RNA World Hypothesis
- Proposed that RNA was the first genetic material.
- RNA could act as both genetic material and an enzyme (ribozymes).
- Over time, DNA evolved as the genetic material because of its stability, and proteins took over enzymatic functions.
Replication of DNA
DNA replication is the process by which DNA makes an identical copy of itself before cell division. It follows a semi-conservative mode, meaning each new DNA molecule has one old strand and one newly synthesized strand.
Experimental Proof of Semi-Conservative Replication (Meselson and Stahl Experiment, 1958)
- Scientists: Matthew Meselson and Franklin Stahl
- Organism Used: E. coli
- Method: Used heavy nitrogen (¹⁵N) and light nitrogen (¹⁴N) to label DNA.
Steps of the Experiment:
- E. coli was grown in a medium containing ¹⁵N (heavy nitrogen) → DNA became heavier.
- These bacteria were then transferred to a ¹⁴N (light nitrogen) medium.
- After one round of replication, DNA was intermediate (hybrid ¹⁵N-¹⁴N).
- After two rounds, DNA separated into half hybrid and half light strands.
Results of the Experiment
- The first generation had intermediate DNA (one heavy and one light strand).
- The second generation had 50% hybrid and 50% light DNA.
- This proved that DNA replication is semi-conservative.
Experiment by Taylor and Colleagues (1958)
- Organism Used: Root cells of Vicia faba (fava bean).
- Method: Used radioactive ³H-thymidine to label DNA.
- Findings: Observed that one strand of the chromosome contained radioactive DNA after the first division, confirming semi-conservative replication in eukaryotes.
Replication Machinery and Enzymes
DNA replication occurs at the replication fork and involves multiple enzymes that help unwind, copy, and seal DNA strands.
Table: Enzymes Involved in DNA Replication and Their Functions
| Enzyme | Function |
|---|---|
| Helicase | Unwinds the DNA helix by breaking hydrogen bonds |
| Topoisomerase | Relieves supercoiling tension by making cuts in DNA strands |
| Single-Strand Binding Proteins (SSBs) | Prevents re-annealing of single strands |
| Primase | Synthesizes RNA primers to initiate replication |
| DNA Polymerase III | Adds nucleotides to the growing DNA strand in a 5’ to 3’ direction |
| DNA Polymerase I | Removes RNA primers and replaces them with DNA |
| Ligase | Seals gaps between Okazaki fragments on the lagging strand |
Transcription
Introduction to Transcription
Transcription is the process of synthesizing RNA from a DNA template. It is the first step of gene expression, where the genetic information in DNA is copied into messenger RNA (mRNA), which carries the instructions for protein synthesis.
Key Points:
- Transcription occurs in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells.
- It is catalyzed by the enzyme RNA polymerase.
- Only one strand of DNA (template strand) is used to synthesize RNA.
- The produced RNA strand is complementary to the template strand and identical to the coding strand (except thymine (T) is replaced by uracil (U)).
Transcription Unit
A transcription unit is the segment of DNA that is transcribed into RNA. It consists of:
- Promoter:
- A DNA sequence located upstream of the gene.
- Acts as the binding site for RNA polymerase.
- Determines the starting point for transcription.
- Structural Gene:
- The actual DNA sequence that codes for the RNA.
- Only one strand (template strand) is transcribed.
- The other strand is the coding strand, which resembles the RNA sequence.
- Terminator:
- A DNA sequence that signals the end of transcription.
- Causes RNA polymerase to detach from the DNA strand.
Transcription Unit and the Gene
A gene is a functional unit of inheritance. It is part of the transcription unit and consists of:
- Exons: Coding sequences that are expressed in the final mRNA.
- Introns: Non-coding sequences that are removed during RNA splicing (only in eukaryotes).
Coding and Template Strands:
- The coding strand (5′ → 3′) carries the same sequence as the RNA (except thymine is replaced with uracil).
- The template strand (3′ → 5′) acts as the template for RNA synthesis.
Types of RNA
There are three main types of RNA involved in transcription and translation:
- Messenger RNA (mRNA):
- Carries the genetic code from DNA to ribosomes.
- Contains codons (triplet base sequences) that specify amino acids.
- Transfer RNA (tRNA):
- Brings specific amino acids to the ribosome.
- Contains an anticodon complementary to the mRNA codon.
- Ribosomal RNA (rRNA):
- Structural and catalytic component of ribosomes.
- Provides the site for protein synthesis.
Process of Transcription
Steps of Transcription:
- Initiation:
- RNA polymerase binds to the promoter region.
- The DNA helix unwinds, and transcription begins.
- Elongation:
- RNA polymerase adds complementary ribonucleotides in the 5′ → 3′ direction.
- Uracil (U) is incorporated instead of thymine (T).
- Termination:
- Transcription stops when RNA polymerase reaches the terminator sequence.
- The newly synthesized RNA is released.
Process of Transcription in Prokaryotes
Transcription in prokaryotes is simpler and occurs in the cytoplasm.
Key Features:
- Single RNA polymerase synthesizes all types of RNA (mRNA, tRNA, and rRNA).
- No introns are present, so no splicing is required.
- Transcription and translation are coupled—both processes occur simultaneously.
- mRNA does not require any processing and is directly used for translation.
Steps:
- Initiation:
- RNA polymerase binds to the promoter region with the help of a sigma (σ) factor.
- The Pribnow box (TATAAT) in the promoter region facilitates RNA polymerase binding.
- Elongation:
- RNA polymerase moves along the template strand, adding nucleotides in the 5′ → 3′ direction.
- Termination:
- Occurs by two mechanisms:
- Rho-dependent termination: Rho protein detaches RNA polymerase.
- Rho-independent termination: Formation of a hairpin loop in the RNA causes detachment.
Process of Transcription in Eukaryotes
Transcription in eukaryotes is more complex and occurs in the nucleus.
Key Features:
- Three types of RNA polymerases:
- RNA Polymerase I: Synthesizes rRNA.
- RNA Polymerase II: Synthesizes mRNA.
- RNA Polymerase III: Synthesizes tRNA and some small RNAs.
- The mRNA undergoes post-transcriptional modifications:
- Capping: Addition of a 7-methylguanosine cap at the 5′ end.
- Polyadenylation: Addition of a poly-A tail at the 3′ end.
- Splicing: Removal of introns by spliceosomes.
- Transcription and translation are separated—transcription occurs in the nucleus, while translation occurs in the cytoplasm.
Steps:
- Initiation:
- Transcription factors help RNA polymerase bind to the promoter.
- The promoter contains a TATA box.
- Elongation:
- RNA polymerase moves along the template strand, synthesizing RNA in the 5′ → 3′ direction.
- Termination:
- RNA polymerase detaches when it reaches a termination signal.
- Post-Transcriptional Modifications:
- Capping, polyadenylation, and splicing make the mRNA mature and ready for translation.
Comparison of Transcription in Prokaryotes and Eukaryotes (Table)
| Feature | Prokaryotic Transcription | Eukaryotic Transcription |
|---|---|---|
| Location | Cytoplasm | Nucleus |
| RNA Polymerase | Single RNA polymerase | Three RNA polymerases (I, II, III) |
| Promoter Sequence | Pribnow box (TATAAT) | TATA box in promoter |
| Initiation Factors | Uses σ factor | Uses multiple transcription factors |
| Post-transcriptional Modifications | Absent | Present (capping, polyadenylation, splicing) |
| Introns and Exons | Absent | Present (introns removed during splicing) |
| Coupling with Translation | Transcription and translation are coupled | Transcription and translation are separate processes |
| mRNA Maturation | No processing required | mRNA undergoes modifications before translation |
Genetic Code
The genetic code is the set of rules by which the information in DNA or RNA is translated into proteins. It determines how sequences of nucleotides specify the amino acids that form proteins.
Key Points about Genetic Code:
- Definition:
- It is a triplet code where three nucleotides (called a codon) specify one amino acid.
- Universal Nature:
- The genetic code is universal in all living organisms (except a few exceptions in mitochondria and some protozoans).
- Degenerate:
- Multiple codons can code for the same amino acid.
- Example: Both GAA and GAG code for glutamic acid.
- Non-Overlapping and Comma-less:
- The code is read continuously from a fixed point in groups of three nucleotides without overlapping.
- Example: AUG-GCA-UUC codes for Met-Ala-Phe, not AUG-GAU-CAU.
- Start and Stop Codons:
- Start codon: AUG → codes for methionine, signaling the beginning of translation.
- Stop codons: UAA, UAG, and UGA → do not code for any amino acid, signaling the end of translation.
- Collinearity:
- The sequence of codons in mRNA corresponds exactly to the sequence of amino acids in the protein.
- Unambiguous:
- Each codon specifies only one amino acid.
- Redundancy:
- Some amino acids are specified by more than one codon.
Mutations and the Genetic Code
Mutation is a change in the nucleotide sequence of DNA that can alter the genetic code and the protein formed.
Types of Mutations:
- Point Mutation:
- A single nucleotide is changed.
- Example: Sickle cell anemia occurs due to a mutation in the β-globin gene where GAG (glutamic acid) is changed to GUG (valine).
- Frameshift Mutation:
- Caused by insertion or deletion of nucleotides.
- It shifts the reading frame, changing the entire amino acid sequence downstream.
- Silent Mutation:
- A change in the codon that does not alter the amino acid.
- Example: GGA (glycine) → GGC (glycine).
- Nonsense Mutation:
- A codon is changed into a stop codon, causing premature termination.
- Example: UAC → UAA (stop codon).
- Missense Mutation:
- A codon change results in a different amino acid.
- Example: GAG (glutamic acid) → GTG (valine).
tRNA: The Adapter Molecule
Transfer RNA (tRNA) is a small RNA molecule that acts as an adapter during translation by linking specific amino acids to the corresponding codons on mRNA.
Structure of tRNA:
- Cloverleaf Structure:
- tRNA has a cloverleaf structure with three key loops:
- D-loop: Helps in recognition by aminoacyl-tRNA synthetase.
- TΨC loop: Binds to the ribosome.
- Anticodon loop: Contains a triplet sequence (anticodon) complementary to the mRNA codon.
- Amino Acid Attachment Site:
- The 3′ end of tRNA carries the specific amino acid.
- Sequence: CCA at the 3’ end.
- Anticodon:
- Recognizes the complementary codon on mRNA during translation.
- Function:
- tRNA brings the correct amino acid to the ribosome based on the mRNA codon sequence.
Translation
Translation is the process of protein synthesis where the mRNA sequence is decoded to form a polypeptide chain. It occurs in the cytoplasm at ribosomes.
Steps of Translation:
1. Initiation:
- The small ribosomal subunit binds to the mRNA at the start codon (AUG).
- The initiator tRNA carrying methionine (Met) binds to the start codon.
- The large ribosomal subunit then binds, forming the complete ribosome.
- The ribosome has three sites:
- A-site (Aminoacyl site) → holds incoming aminoacyl-tRNA.
- P-site (Peptidyl site) → holds the growing polypeptide chain.
- E-site (Exit site) → tRNA exits the ribosome.
2. Elongation:
- Codon recognition: tRNA with the complementary anticodon binds to the mRNA codon at the A-site.
- Peptide bond formation: A peptide bond forms between the amino acids at the P-site and A-site.
- The ribosome moves along the mRNA in the 5′ → 3′ direction.
- The empty tRNA exits from the E-site.
- The growing polypeptide chain is transferred from the tRNA in the P-site to the tRNA in the A-site.
3. Termination:
- When the ribosome reaches a stop codon (UAA, UAG, or UGA), the translation process stops.
- Release factors bind to the ribosome, causing the release of the polypeptide chain.
- The ribosome dissociates, completing the translation process.
Key Points of Translation:
- mRNA carries the genetic code from DNA.
- tRNA acts as the adapter molecule, bringing amino acids.
- The ribosome is the site of translation.
- The polypeptide chain grows as codons are read sequentially.
- Proteins formed undergo post-translational modifications before becoming functional.
Important Details for NEET:
- Wobble Hypothesis: The third base of the codon has flexible pairing rules, allowing non-standard base pairing.
- Polysome (Polyribosome): When multiple ribosomes translate a single mRNA simultaneously.
- Post-Translational Modifications: Proteins may undergo modifications such as folding, cleavage, and addition of functional groups.
Regulation of Gene Expression
Gene expression refers to the process by which genetic information is used to synthesize proteins. Regulation of gene expression is crucial for controlling cellular functions, responding to environmental changes, and ensuring proper development.
Gene Regulation at the Translational Level
Gene expression can be regulated at multiple levels, including:
- Transcriptional Level: Regulation during the process of transcription.
- Post-Transcriptional Level: Regulation after the formation of mRNA (splicing, capping, etc.).
- Translational Level: Regulation during protein synthesis from mRNA.
- Post-Translational Level: Regulation after the protein is formed.
Translational Level Regulation:
- Definition:
- Involves controlling the efficiency of mRNA translation into proteins.
- Mechanisms:
- Regulatory Proteins: Bind to mRNA and either enhance or inhibit translation.
- MicroRNA (miRNA) and siRNA:
- Small RNA molecules that bind to complementary mRNA.
- Prevent translation by degrading mRNA or blocking ribosome attachment.
- Riboswitches:
- RNA sequences that change conformation when bound to specific molecules, affecting translation efficiency.
- mRNA Stability:
- The longer an mRNA molecule remains intact, the more proteins it can produce.
- mRNA stability is regulated by RNA-binding proteins.
Lac Operon Mechanism of Regulation
The lac operon is a classic model of gene regulation in prokaryotes, specifically in E. coli. It regulates the metabolism of lactose.
Components of the Lac Operon:
- Structural Genes:
- lacZ: Codes for β-galactosidase, which breaks down lactose into glucose and galactose.
- lacY: Codes for permease, which allows lactose entry into the cell.
- lacA: Codes for transacetylase, whose function is not fully understood.
- Promoter (P):
- Site where RNA polymerase binds to initiate transcription.
- Operator (O):
- A DNA sequence where the repressor protein binds, blocking transcription.
- Regulator Gene (lacI):
- Codes for the repressor protein that binds to the operator and inhibits transcription.
Mechanism of Regulation:
1. In the Absence of Lactose:
- The repressor protein (produced by lacI) binds to the operator.
- This prevents RNA polymerase from transcribing the structural genes.
- The operon remains switched off.
2. In the Presence of Lactose:
- Lactose acts as an inducer by binding to the repressor protein.
- This causes a conformational change, making the repressor inactive.
- RNA polymerase can now bind to the promoter and transcribe the structural genes.
- The enzymes required for lactose metabolism are produced.
Significance of Lac Operon:
- Demonstrates gene regulation by feedback inhibition.
- Helps conserve energy by only expressing the genes when lactose is present.
Human Genome Project (HGP)
The Human Genome Project (HGP) was an international scientific effort launched in 1990 and completed in 2003 to sequence the entire human genome.
Goals of the Human Genome Project:
- Map all human genes and their location on chromosomes.
- Determine the complete nucleotide sequence of the human genome.
- Identify and map all the genes associated with diseases.
- Store genetic information in databases for public access.
- Improve tools for data analysis.
- Address the ethical, legal, and social issues (ELSI) associated with genome research.
Methodologies of HGP:
The project involved two main approaches:
- Expressed Sequence Tags (ESTs):
- Focused on identifying and sequencing only the expressed genes (coding regions).
- Sequence Annotation:
- Sequencing the entire genome, including both coding and non-coding regions.
Steps Involved in Sequencing:
- Isolation of DNA:
- DNA is extracted from cells.
- Fragmentation:
- The DNA is broken into smaller fragments using restriction enzymes.
- Cloning:
- Fragments are cloned into vectors (bacterial or yeast artificial chromosomes).
- Sequencing:
- The nucleotide sequences of the DNA fragments are determined using automated sequencing machines.
- Alignment:
- The sequences are aligned using bioinformatics tools.
- Annotation:
- The identified sequences are matched with known genes.
Salient Features of the Human Genome:
- Total Genes:
- The human genome contains approximately 20,000–25,000 genes.
- Base Pairs:
- Contains 3.2 billion base pairs.
- Protein-Coding Genes:
- Only 1.5% of the genome codes for proteins.
- Repetitive Sequences:
- Nearly 50% of the genome consists of repetitive sequences.
- Gene Distribution:
- Genes are unevenly distributed across chromosomes.
- Junk DNA:
- Large portions of the genome consist of non-coding DNA, previously considered “junk” but now known to have regulatory functions.
- Variations:
- Single nucleotide polymorphisms (SNPs) account for genetic variations.
- Mitochondrial DNA:
- Mitochondrial DNA contains 37 genes involved in cellular respiration.
DNA Fingerprinting
DNA fingerprinting is a technique used to identify individuals by analyzing their unique DNA sequences. It was developed by Alec Jeffreys in 1985.
Mechanism of DNA Fingerprinting:
- DNA Extraction:
- DNA is extracted from cells (blood, hair, or saliva).
- DNA Digestion:
- DNA is cut into fragments using restriction enzymes.
- Gel Electrophoresis:
- The DNA fragments are separated by size using gel electrophoresis.
- Southern Blotting:
- The separated DNA fragments are transferred onto a nylon membrane.
- Hybridization:
- The membrane is treated with radioactive or fluorescent probes that bind to specific DNA sequences.
- Detection:
- The DNA pattern is visualized using X-ray film or UV light.
- Comparison:
- The DNA profile is compared with reference samples.
Applications of DNA Fingerprinting:
- Forensic Science:
- Used to identify criminals by matching DNA from crime scenes.
- Paternity Testing:
- Determines biological parentage.
- Identification of Victims:
- Helps identify victims of accidents or natural disasters.
- Disease Diagnosis:
- Used to detect genetic disorders.
- Evolutionary Studies:
- Used to determine genetic relationships among species.
- Personalized Medicine:
- Helps in identifying genetic predisposition to diseases.