Molecular Basis of Inheritance Flashcards
(98 cards)
1
Q
What is the total length of DNA in a mammalian cell, and how is it calculated?
A
The length of DNA in a typical mammalian cell is about 2.2 meters. It is calculated using:
Total DNA base pairs × Distance between consecutive base pairs
6.6 × 10⁹ bp × 0.34 × 10⁻⁹ m/bp = 2.2 m
2
Q
How does the length of DNA compare to the size of the nucleus?
A
The nucleus is only 10⁻⁶ m in diameter, meaning DNA is about a million times longer than the nucleus! This makes DNA packaging essential for fitting into the nucleus.
3
Q
How can we calculate the number of base pairs in E. coli DNA?
A
Given that the length of E. coli DNA is 1.36 mm (1.36 × 10⁻³ m), and using the same formula:
Number of base pairs = DNA length / Distance between base pairs
= (1.36 × 10⁻³ m) / (0.34 × 10⁻⁹ m/bp) ≈ 4.0 × 10⁶ bp
4
Q
How is DNA packaged in prokaryotes?
A
- Prokaryotes lack a nucleus, but their DNA is not freely floating.
- DNA is held in a region called the nucleoid, where it is supercoiled into loops by positively charged proteins that bind to the negatively charged DNA.
5
Q
What are histones, and why are they important in DNA packaging?
A
- Histones are positively charged proteins rich in lysine and arginine (which have positive side chains).
- Their positive charge allows them to bind to negatively charged DNA and package it efficiently.
6
Q
What is a histone octamer?
A
- A histone octamer is a complex of eight histone proteins.
- DNA wraps around this octamer to form a nucleosome, which is the basic unit of DNA packaging.
7
Q
What is a nucleosome, and how many base pairs does it contain?
A
- A nucleosome consists of DNA wrapped around a histone octamer.
- Each nucleosome contains 200 base pairs of DNA.
- When viewed under an electron microscope, nucleosomes appear as ‘beads on a string’.
8
Q
How is chromatin further packaged?
A
- Nucleosomes form the ‘beads-on-string’ structure.
- These nucleosomes coil to form chromatin fibers.
- During metaphase (cell division), chromatin fibers condense to form chromosomes.
9
Q
What are Non-Histone Chromosomal (NHC) proteins?
A
- NHC proteins help in higher-order chromatin packaging.
- They are needed to supercoil DNA beyond nucleosomes and help form chromosomes.
10
Q
What are euchromatin and heterochromatin?
A
- Euchromatin is loosely packed, stains light, and is transcriptionally active.
- Heterochromatin is tightly packed, stains dark, and is transcriptionally inactive.
11
Q
Why did it take so long to discover that DNA is the genetic material?
A
- Even though Mendel’s principles of inheritance and Meischer’s discovery of nuclein occurred around the same time, it took decades to prove DNA’s role.
- By 1926, scientists had determined that genetic material was on chromosomes, but they did not know whether it was DNA or protein.
12
Q
What was Griffith’s experiment, and what did it show?
A
- Frederick Griffith (1928) experimented with Streptococcus pneumoniae, a bacterium that causes pneumonia.
- He found two strains:
S strain (smooth, virulent) → Has a mucous (polysaccharide) coat and causes disease.
R strain (rough, non-virulent) → Lacks the coat and does not cause disease. - Key Observations:
1. Live S strain → Killed mice ✅
2. Live R strain → Mice survived ❌
3. Heat-killed S strain → Mice survived ❌
4. Heat-killed S strain + Live R strain → Mice died! ✅ - He found living S strain bacteria in the dead mice, proving that the R strain had transformed into S strain.
13
Q
What was Griffith’s conclusion?
A
- R strain bacteria were ‘transformed’ into the virulent S strain.
- Some ‘transforming principle’ from the heat-killed S strain was responsible for this change.
- This principle carried genetic information, but Griffith did not identify it as DNA.
14
Q
What is the significance of Griffith’s experiment?
A
- It introduced the idea of genetic transformation—one organism can pass genetic traits to another.
- However, Griffith did not identify DNA as the genetic material, which was later determined by Avery, MacLeod, and McCarty.
15
Q
Who confirmed the biochemical nature of the transforming principle?
A
- Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-1944).
16
Q
How did Avery, MacLeod, and McCarty determine the transforming principle?
A
- They purified different biomolecules (proteins, DNA, RNA) from heat-killed S strain and tested their ability to transform R strain into S strain.
- Key findings:
1. DNA alone from S strain could transform R strain into S strain.
2. Protease (protein-digesting enzyme) and RNase (RNA-digesting enzyme) had no effect, meaning the transforming principle was not protein or RNA.
3. DNase (DNA-digesting enzyme) stopped transformation, proving that DNA is the genetic material.
17
Q
What was the conclusion of Avery, MacLeod, and McCarty’s experiment?
A
- DNA, not protein, is the genetic material responsible for transformation.
- However, not all biologists were convinced at the time.
18
Q
What is the difference between DNA and DNase?
A
- DNA is the genetic material that carries hereditary information.
- DNase is an enzyme that breaks down DNA, preventing it from functioning.
19
Q
Question
A
Answer
20
Q
Who provided the ultimate proof that DNA is the genetic material?
A
- Alfred Hershey and Martha Chase (1952).
21
Q
What organism did Hershey and Chase work with?
A
- They worked with bacteriophages, which are viruses that infect bacteria.
22
Q
What was the goal of Hershey and Chase’s experiment?
A
- To determine whether DNA or protein was the genetic material passed from a bacteriophage (virus) to its bacterial host (E. coli).
23
Q
How does a bacteriophage infect a bacterial cell?
A
- The bacteriophage attaches to the bacterial cell and injects its genetic material inside.
- The bacterial cell then treats the viral genetic material as its own, producing more virus particles.
24
Q
What was the key question in their experiment?
A
- Scientists knew that bacteriophages transferred their genetic material to bacteria.
- The question was: Is the genetic material being transferred DNA or protein?
25
How did Hershey and Chase label DNA and proteins in their experiment?
- They grew bacteriophages in two different radioactive media:
1. Radioactive Phosphorus (³²P): Labeled DNA because DNA contains phosphorus.
2. Radioactive Sulfur (³⁵S): Labeled protein because proteins contain sulfur but DNA does not.
26
What did they do after labeling the DNA and protein?
- Radioactive phages were allowed to infect E. coli bacteria.
- After infection, the viral coats (capsids) were removed from bacteria using a blender.
- The mixture was then spun in a centrifuge to separate the virus particles from bacterial cells.
27
What were the key observations of Hershey and Chase’s experiment?
1. Bacteria infected with phages that had radioactive DNA (³²P) were radioactive. ✅
- This means DNA entered the bacterial cells.
2. Bacteria infected with phages that had radioactive protein (³⁵S) were NOT radioactive. ❌
- This means proteins did not enter the bacterial cells.
28
What was the conclusion of Hershey and Chase’s experiment?
- DNA, not protein, is the genetic material.
- The DNA of the virus enters the bacterial cell and directs the production of new viruses.
29
Why was Hershey and Chase’s experiment important?
- It provided the final proof that DNA carries genetic information.
- It ended the debate about whether DNA or protein was the genetic material.
30
Criteria for a Molecule to be Genetic Material
- A genetic material must fulfill these four criteria: 1. Replication – Must be able to make copies of itself. 2. Stability – Should be chemically and structurally stable. 3. Mutation & Evolution – Must allow slow changes for evolution. 4. Expression – Should express traits as per Mendelian inheritance. - DNA & RNA both satisfy these, but proteins fail at replication.
31
Why DNA is the Predominant Genetic Material?
- Stability: DNA is chemically and structurally more stable than RNA. - Complementary Base Pairing: Allows DNA to maintain integrity even after heat denaturation (as seen in Griffith’s experiment). - Lack of 2’-OH in DNA: RNA has a reactive 2’-OH group, making it more labile and degradable, while DNA lacks this, increasing stability. - Thymine vs Uracil: DNA contains thymine instead of uracil, which enhances stability and reduces errors.
32
Comparison: DNA vs RNA as Genetic Material
- Mutation Rate: RNA mutates faster than DNA (useful for viral evolution). - Expression: RNA directly codes for proteins, while DNA requires RNA as an intermediate. - Evolutionary Preference: DNA is more suited for long-term storage of genetic information, while RNA is better for quick transmission (e.g., mRNA in protein synthesis). - Examples: DNA is the genetic material in most organisms, while some viruses (e.g., TMV, QB bacteriophage) use RNA.
33
The RNA World Hypothesis
- RNA was the first genetic material, as supported by evidence from biochemical evolution. - Roles of RNA in early life: 1. Genetic Material – Stored and transmitted genetic information. 2. Catalyst – Some RNA molecules function as enzymes (ribozymes). 3. Metabolism & Translation – RNA played a key role in early biochemical reactions. - Why DNA evolved from RNA? 1. RNA is chemically unstable and highly reactive. 2. DNA evolved by chemical modifications, making it more stable. 3. DNA’s double-stranded nature provided a repair mechanism, reducing errors.
34
Watson & Crick’s Model of DNA Replication
- Proposed semiconservative replication based on the DNA double-helix model. - Process: 1. The two DNA strands separate and act as templates. 2. Complementary base pairing guides the synthesis of new strands. 3. Each new DNA molecule consists of one parental strand and one newly synthesized strand. - Quote by Watson & Crick (1953): “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
35
Meselson & Stahl Experiment (Proof of Semiconservative DNA Replication)
- Experiment with E. coli (1958): 1. Grew E. coli in a medium containing 15NH₄Cl (heavy nitrogen). 2. 15N was incorporated into newly synthesized DNA. 3. Transferred bacteria to 14NH₄Cl (normal nitrogen) and allowed replication. - Observations (Using CsCl Density Gradient Centrifugation): 1. After one generation (20 min) – All DNA was hybrid (15N-14N). 2. After two generations (40 min) – Half hybrid (15N-14N), half light (14N-14N). 3. After more generations – Increasing proportion of light DNA. - Conclusion: DNA replicates semiconservatively, proven in both bacteria and eukaryotes (Taylor’s experiment on Vicia faba by radioactive thymidine).
36
Enzymes Involved in DNA Replication
- Main enzyme: DNA-dependent DNA polymerase – Uses a DNA template to polymerize deoxynucleotides. - Properties of DNA Polymerase: 1. Highly efficient – Must replicate millions of base pairs quickly. 2. Highly accurate – Errors can cause mutations. 3. Energetically expensive – Uses deoxyribonucleoside triphosphates (dNTPs) as both substrates and energy sources. - Additional enzymes required for replication: 1. Helicase – Unwinds the DNA double helix. 2. Topoisomerase – Prevents DNA supercoiling. 3. Primase – Synthesizes RNA primers. 4. DNA Ligase – Joins Okazaki fragments on the lagging strand.
37
Mechanism of DNA Replication
- Replication occurs at the Replication Fork: 1. DNA strands cannot fully separate due to high energy requirements. 2. A small opening in the DNA helix is created where replication occurs. - Direction of Replication: 1. Leading Strand (3'→5' Template): Continuous synthesis by DNA polymerase in 5'→3' direction. 2. Lagging Strand (5'→3' Template): Synthesized in Okazaki fragments, later joined by DNA ligase.
38
Origin of Replication & Cell Cycle Coordination
- Replication begins at a specific site – the origin of replication. - In E. coli, replication starts at a single origin, while eukaryotic cells have multiple origins. - Importance in recombinant DNA technology: 1. Vectors (plasmids, phages, etc.) must contain an origin of replication for DNA propagation. - Replication is tightly regulated with the Cell Cycle: 1. Occurs in the S-phase of the eukaryotic cell cycle. 2. Failure in cell division after replication leads to polyploidy (extra chromosome sets).
39
What is Transcription?
- Definition: Transcription is the process of copying genetic information from one strand of DNA into RNA. - Key principle: Complementarity, except A pairs with U (uracil) instead of T. - Differences from DNA Replication: 1. Only a segment of DNA is copied, not the entire genome. 2. Only one strand is transcribed, avoiding issues like: - Producing different proteins from the same gene. - Formation of double-stranded RNA, which would prevent translation.
40
The Transcription Unit & Its Components
- A transcription unit consists of: 1. Promoter – Located upstream (5' end), binds RNA polymerase and defines template & coding strands. 2. Structural Gene – The actual sequence transcribed into RNA. 3. Terminator – Located downstream (3' end), marks the stop point for transcription. - Strands of DNA in Transcription: 1. Template Strand (3'→5') – Used for RNA synthesis. 2. Coding Strand (5'→3') – Has the same sequence as RNA (except T → U). - Important Note: Switching the positions of the promoter and terminator can reverse the definition of coding and template strands.
41
RNA Sequence Transcription Example
- Given DNA sequences: Template Strand: 3'-ATGCATGCATGCATGCATGCATGC-5' Coding Strand: 5'-TACGTACGTACGTACGTACGTACG-3' - Transcribed RNA Sequence: 5'-UACGUACGUACGUACGUACGUACG-3'
42
Transcription Unit & Gene Definition
- Gene: Functional unit of inheritance, but defining it precisely is difficult. - Cistron: Segment of DNA coding for a polypeptide. - Types of Structural Genes: 1. Monocistronic (Eukaryotes) – Codes for a single protein. 2. Polycistronic (Prokaryotes) – Codes for multiple proteins. - Split Genes in Eukaryotes: - Exons: Coding sequences appearing in mature RNA. - Introns: Non-coding sequences removed during processing. - Regulatory Sequences: Influence gene expression but do not code for RNA or protein. Sometimes termed regulatory genes.
43
Types of RNA and Their Roles
- mRNA (Messenger RNA) – Provides the template for protein synthesis. - tRNA (Transfer RNA) – Brings amino acids and reads the genetic code. - rRNA (Ribosomal RNA) – Plays structural & catalytic roles in translation.
44
Transcription Process in Bacteria
- Single RNA Polymerase catalyzes all types of RNA synthesis. - Steps in Transcription: 1. Initiation – RNA polymerase binds to the promoter with the help of σ-factor. 2. Elongation – Polymerase moves along DNA, synthesizing RNA. 3. Termination – ρ-factor helps in stopping transcription, releasing RNA. - Key Features in Prokaryotes: 1. No need for RNA processing (mRNA is functional immediately). 2. Transcription & translation occur simultaneously in the cytoplasm.
45
Transcription in Eukaryotes: Complexities & RNA Processing
- Three RNA Polymerases in Nucleus: 1. RNA Polymerase I – Transcribes rRNA (28S, 18S, 5.8S). 2. RNA Polymerase II – Transcribes precursor mRNA (hnRNA). 3. RNA Polymerase III – Transcribes tRNA, 5S rRNA, and snRNA. - Processing of hnRNA (Precursor mRNA): 1. Splicing – Introns removed, exons joined in a defined order. 2. Capping – Methyl guanosine triphosphate added to 5'-end. 3. Tailing – 200-300 adenylate residues added to 3'-end. - Final Product: Fully processed mRNA, transported for translation.
46
Significance of Eukaryotic RNA Processing
- Split-gene arrangement is an ancient feature of the genome. - Introns are thought to be remnants of the early "RNA world". - Splicing and RNA-dependent processes are increasingly important in genetic regulation and evolution.
47
Concept of Genetic Code
- Replication & Transcription involve nucleotide copying (complementarity). - Translation transfers genetic info from nucleotides → amino acids (no complementarity exists). - Changes in nucleic acids → changes in proteins, proving a genetic code exists. - George Gamow (Physicist): Proposed that three nucleotides (triplet) form a codon to code for one amino acid. - Har Gobind Khorana: Synthesized defined RNA sequences. - Marshall Nirenberg: Developed a cell-free system for protein synthesis, helping decode the genetic code. - Severo Ochoa: Used polynucleotide phosphorylase to polymerize RNA without a template.
48
Features of the Genetic Code
1. Triplet Nature – 3 nucleotides form a codon. 2. Degenerate Code – More than one codon codes for some amino acids. 3. Non-overlapping & Continuous – Read without punctuation. 4. Universal – Same in most organisms (e.g., UUU = Phenylalanine in both bacteria & humans). Exceptions: Some mitochondrial codons, protozoans. 5. AUG (Start Codon) – Codes for Methionine and acts as initiator codon. 6. Stop Codons – UAA, UAG, UGA (Terminate translation).
49
Deciphering a Genetic Code Example
mRNA sequence: AUG UUU UUC UUC UUU UUU UUC - AUG → Methionine (Start Codon) - UUU → Phenylalanine - UUC → Phenylalanine - Final sequence: Met-Phe-Phe-Phe-Phe-Phe-Phe Reverse Prediction Issue: - Multiple mRNA sequences can code for the same amino acid sequence due to degeneracy.
50
Mutations & Genetic Code
- Mutation: A change in DNA sequence that affects gene function. - Point Mutation Example: Sickle Cell Anemia - Cause: Single base change in beta-globin gene (Glutamate → Valine). - Frameshift Mutations: - Insertion/Deletion of 1 or 2 bases → Alters reading frame (Drastic change). - Insertion/Deletion of 3 bases (or multiples of 3) → Adds/deletes whole codons, but the reading frame remains intact. Example: - Normal: RAM HAS RED CAP - Insert 'B': RAM HAS BRE DCA P - Insert 'BIG': RAM HAS BIG RED CAP (Frame remains correct). - Delete ‘R’: RAM HAS EDC AP (Shifts frame, making nonsense words).
51
tRNA – The Adapter Molecule
- Francis Crick's Adapter Hypothesis: Proposed that an intermediate molecule must read the genetic code and link it to amino acids (since amino acids cannot directly read codons). - tRNA (Transfer RNA): Initially called sRNA (soluble RNA), later identified as the adapter molecule. - Structure of tRNA: 1. Anticodon Loop: Contains bases complementary to the mRNA codon. 2. Amino Acid Acceptor End: Binds to a specific amino acid. 3. Clover-Leaf Model (Secondary Structure): Actual 3D shape is inverted L. - Specificity: Each tRNA is specific for a particular amino acid. - Initiator tRNA: Special tRNA that recognizes the start codon (AUG) and begins translation. - No tRNAs exist for stop codons.
52
Translation – Protein Synthesis
- Definition: Process of polymerizing amino acids into a polypeptide based on the mRNA sequence. - Peptide Bond Formation: Requires energy and occurs between adjacent amino acids. - Steps of Translation: 1. Aminoacylation of tRNA (Charging of tRNA): - Amino acids are activated using ATP and linked to their cognate tRNA. - Ensures correct amino acid is incorporated into the protein. 2. Ribosome – The Cellular Factory: - Made of structural RNAs + ~80 proteins. - Two subunits: Large & Small (exist separately in inactive form). - 23S rRNA (Bacteria) functions as a ribozyme to catalyze peptide bond formation.
53
Translational Unit & mRNA Structure
- mRNA components: 1. Start codon (AUG): Initiates translation. 2. Coding sequence: Specifies amino acid sequence. 3. Stop codon (UAA, UAG, UGA): Terminates translation. - Untranslated Regions (UTRs): - Found at 5' (before start codon) & 3' (after stop codon) ends. - Aid in efficient translation but do not code for proteins.
54
Steps of Translation
1. Initiation: - Small ribosomal subunit binds to mRNA at AUG (start codon). - Initiator tRNA recognizes AUG and binds. - Large ribosomal subunit joins, forming the complete ribosome. 2. Elongation: - Charged tRNAs bring amino acids to the ribosome. - Each tRNA binds to its complementary codon in mRNA. - Ribosome moves codon by codon, catalyzing peptide bond formation between amino acids. 3. Termination: - A release factor binds when a stop codon is reached. - The completed polypeptide is released from the ribosome.
55
Regulation of Gene Expression in eukaryotes
Definition: Control of gene activity to ensure proteins are produced only when required, preventing energy wastage.
🔹 Regulation Levels in Eukaryotes:
Transcriptional Regulation: Control over RNA polymerase binding & transcription initiation.
Post-Transcriptional Regulation: Control over mRNA processing (splicing, capping, polyadenylation, stability).
mRNA Transport Control: Regulation of mRNA exit from nucleus to cytoplasm.
Translational Regulation: Controlling efficiency of ribosome binding to mRNA.
🔹 Why is Gene Regulation Important?
Metabolic Adaptation: Genes like β-galactosidase in E. coli are only expressed when needed (e.g., presence of lactose).
Development & Differentiation: Different genes are switched on/off in embryogenesis & organ development.
Environmental Responses: Regulation allows organisms to adapt to external stimuli.
56
Gene Regulation in Prokaryotes
Primary Control Point: Transcriptional initiation (mainly regulates gene expression in bacteria).
🔹 Key Players in Transcription Regulation:
Promoter: The binding site for RNA polymerase to start transcription.
Operator: A regulatory DNA sequence where a repressor binds to block transcription.
Regulatory Proteins:
Activators: Enhance RNA polymerase binding (positive regulation).
Repressors: Block RNA polymerase by binding to the operator (negative regulation).
🔹 Operon System: A cluster of functionally related genes controlled by a single promoter & operator.
Examples: Lac operon, Trp operon, Ara operon, His operon, Val operon.
57
The Lac Operon – A Model for Gene Regulation
Discovered by: François Jacob & Jacques Monod.
🔹 Definition: A gene regulatory system in E. coli that controls the breakdown of lactose.
🔹 Components of Lac Operon:
Regulatory Gene (i gene) → Produces repressor protein. i gene-inhibitor gene
Structural Genes:
z gene → Codes for β-galactosidase, breaking lactose into glucose + galactose.
y gene → Codes for permease, increasing lactose entry into the cell.
a gene → Codes for transacetylase, function unclear.
🔹 Why is Lactose the Inducer?
Lactose binds to the repressor, inactivating it, thus allowing gene expression.
Glucose or galactose CANNOT act as inducers.
58
Mechanism of Lac Operon Regulation
In the Absence of Lactose (Operon OFF):
i gene produces a repressor protein.
The repressor binds to the operator, blocking RNA polymerase.
No transcription occurs, NO β-galactosidase, permease, or transacetylase produced.
🟢 In the Presence of Lactose (Operon ON):
Lactose acts as an inducer, binding to the repressor.
The repressor changes shape and detaches from the operator.
RNA polymerase binds to the promoter and starts transcribing z, y, and a genes.
Result: Enzymes for lactose metabolism are produced.
🔹 Important Note: A low level of operon expression always exists to allow lactose to enter the cell initially.
59
Regulation of Lac Operon is negative regulation
Negative Regulation (Repressor-Controlled):
The default state is OFF due to a repressor blocking transcription.
Lactose inactivates the repressor, allowing transcription.
60
Introduction to the Human Genome Project (HGP)
- The genetic makeup of an individual is determined by their DNA sequences. - Differences between individuals arise due to variations in DNA sequences. - This led to the idea of sequencing the entire human genome to understand genetic variation. - The project was launched in 1990 with advancements in genetic engineering and DNA sequencing techniques.
61
HGP – A Mega Project
- The human genome has approximately 3 × 10⁹ base pairs (bp). - Estimated cost at the beginning: $3 per base pair, leading to a total of $9 billion. - If stored in books: - Each page → 1000 letters. - Each book → 1000 pages. - Total books needed → 3300 books for one human genome sequence. - Due to enormous data, high-speed computational devices were required for data storage, retrieval, and analysis. - The HGP was closely linked with the development of Bioinformatics.
62
Goals of the Human Genome Project
1. Identify all human genes (~20,000–25,000 genes). 2. Determine the sequence of all 3 billion base pairs in human DNA. 3. Store DNA sequence data in databases. 4. Develop better tools for analyzing genetic data. 5. Transfer genomic technologies to industries. 6. Address ethical, legal, and social issues (ELSI) related to genetic research.
63
HGP – International Collaboration & Completion
- The Human Genome Project was a 13-year project. - Coordinated by U.S. Department of Energy and National Institutes of Health (NIH). - Major partner: Wellcome Trust (U.K.). - Contributions from Japan, France, Germany, China, and others. - The project was completed in 2003. - The final chromosome (Chromosome 1) was sequenced in May 2006.
64
Significance of HGP
- Understanding DNA variations can lead to new ways to diagnose, treat, and prevent genetic disorders. - Helps in understanding human biology at the molecular level. - Sequencing of non-human organisms aids in: 1. Medical research (e.g., model organisms like E. coli, yeast). 2. Agriculture (e.g., improving crop genomes like rice and Arabidopsis). 3. Energy production. 4. Environmental remediation.
65
Methodologies Used in HGP
- Two Major Approaches: 1. Expressed Sequence Tags (ESTs): - Focused on identifying only the genes that are expressed as RNA. 2. Whole Genome Sequencing: - Sequenced the entire genome (both coding & non-coding regions). - Later assigned functions to different regions (Sequence Annotation).
66
Steps in Whole Genome Sequencing
1. Isolation of total DNA from a human cell. 2. Fragmentation of DNA into smaller pieces (due to technical sequencing limitations). 3. Cloning of fragments into specialized vectors. - Common vectors: - BAC (Bacterial Artificial Chromosome) - YAC (Yeast Artificial Chromosome) - Common hosts: - Bacteria & Yeast. 4. DNA Sequencing using automated DNA sequencers. - Based on Frederick Sanger’s method (credited for amino acids sequencing in proteins). 5. Arranging DNA sequences based on overlapping regions. - Computers & bioinformatics tools were essential for this. 6. Assigning DNA sequences to each chromosome.
67
Final Challenges in HGP
- Chromosome 1 was the last human chromosome to be sequenced in May 2006. - Genetic & Physical Mapping of the genome was crucial. - Required polymorphism data (variation in DNA sequences). - Used restriction endonuclease recognition sites & microsatellite DNA (explained further in DNA Fingerprinting).
68
Examples of Sequenced Non-Human Model Organisms
Bacteria – Used for studying genetic regulation and biotechnology applications. - Yeast – A key model for cell cycle and genetics research. - Caenorhabditis elegans (C. elegans) – A free-living, non-pathogenic nematode, widely used for studying development and aging. - Drosophila melanogaster (Fruit Fly) – A model for genetic studies and developmental biology. - Plants (Rice and Arabidopsis) – Important for plant genetics, crop improvement, and evolutionary studies.
69
Salient Features of the Human Genome
- The Human Genome Project (HGP) revealed several key observations about the human genome.
70
Total Size of the Human Genome
- The human genome consists of 3,164.7 million base pairs (bp).
71
Average Gene Size
- The average gene consists of 3000 bases. - Gene size varies greatly. - The largest known human gene is dystrophin, which spans 2.4 million bases.
72
Total Number of Genes
- Estimated at 30,000 genes. - This is much lower than previous estimates (80,000 – 140,000 genes). - 99.9% of nucleotide bases are identical in all humans.
73
Unknown Gene Functions
- The functions of over 50% of the discovered genes remain unknown.
74
Protein-Coding Region
- Less than 2% of the human genome codes for proteins. - The majority of the genome consists of non-coding sequences.
75
Repeated Sequences in the Genome
- Large portions of the human genome consist of repeated sequences.
76
Repetitive DNA Sequences
- These are stretches of DNA repeated hundreds to thousands of times. - They do not directly code for proteins but provide insights into chromosome structure, dynamics, and evolution.
77
Genes Distribution Across Chromosomes
- Chromosome 1 has the highest number of genes (2968 genes). - Y chromosome has the fewest genes (231 genes).
78
Single Nucleotide Polymorphisms (SNPs)
- Scientists identified 1.4 million locations of single-base DNA variations called SNPs (Single Nucleotide Polymorphisms, ‘snips’). - SNPs are important for: 1. Locating disease-associated genes. 2. Tracing human evolutionary history.
79
Applications and Future Challenges of HGP
- The full understanding of DNA sequences will guide biological research in the coming decades.
80
Interdisciplinary Approach to Genomic Research
- Requires the collaboration of scientists from various fields in both public and private sectors worldwide.
81
New Era of Genomic Research
- The availability of whole-genome sequences enables a new systematic approach to biological research. - Past Research: Scientists studied one or a few genes at a time. - Now: Using high-throughput sequencing technologies, scientists can: 1. Study all genes in a genome. 2. Analyze all transcripts in a specific tissue, organ, or tumor. 3. Investigate how tens of thousands of genes and proteins interact in complex networks to regulate biological functions.
82
DNA Fingerprinting – Definition & Importance
- DNA fingerprinting is a technique used to compare DNA sequences of individuals quickly. - Although 99.9% of DNA is identical in humans, 0.1% differences (around 3 million base pairs) make every individual unique. - It is widely used in forensics, paternity testing, and genetic research.
83
Basis of DNA Fingerprinting
- DNA fingerprinting is based on repetitive DNA sequences, also called satellite DNA. - These sequences show high polymorphism but do not code for proteins. - They are inherited and can be found in any tissue (blood, hair, skin, bone, saliva, sperm).
84
Types of Satellite DNA
- Micro-satellites & Mini-satellites: Differ in length and number of repeats. - They are A:T or G:C rich and do not affect gene function but are crucial for chromosome structure and evolution.
85
Polymorphism & Its Role
- DNA polymorphism arises due to mutations. - If a mutation appears in >1% of the population, it is considered polymorphic. - Polymorphisms in non-coding DNA are more frequent and help in tracing inheritance patterns.
86
Steps of DNA Fingerprinting (Developed by Alec Jeffreys)
1. Isolation of DNA – Extracted from cells. 2. Digestion of DNA – Cut by restriction enzymes. 3. Separation of DNA fragments – Done via gel electrophoresis. 4. Transfer to synthetic membrane – Using Southern blotting. 5. Hybridisation – Use of VNTR (Variable Number Tandem Repeats) probes. 6. Detection – Autoradiography shows unique band patterns.
87
VNTRs (Variable Number Tandem Repeats)
- Mini-satellite DNA with high polymorphism. - The number of repeats varies in each individual, creating a unique DNA profile. - Except identical twins, each individual has a unique pattern.
88
Advancements – PCR & Sensitivity
- PCR (Polymerase Chain Reaction) allows DNA fingerprinting with DNA from a single cell. - More sensitive techniques have improved forensic applications.
89
Applications of DNA Fingerprinting
- Forensic Science – Criminal identification from biological samples. - Paternity & Kinship Testing – Establishing biological relationships. - Genetic Disease Mapping – Identifying inheritable disorders. - Evolutionary Studies – Understanding genetic variations and ancestry.
90
Nucleic Acids – Structure & Function
- Nucleic acids are long polymers of nucleotides. - DNA stores genetic information, while RNA helps in transfer and expression of information. - DNA is more stable than RNA, making it a better genetic material. - RNA evolved first, and DNA was derived from RNA.
91
DNA Structure & Complementary Base Pairing
- DNA is a double-stranded helix, stabilized by hydrogen bonding. - Adenine (A) pairs with Thymine (T) via two H-bonds. - Guanine (G) pairs with Cytosine (C) via three H-bonds. - The two strands are complementary to each other.
92
DNA Replication & Gene Definition
- DNA replicates semiconservatively, where each new strand is guided by complementary base pairing. - A gene is a segment of DNA that codes for RNA. - During transcription, one DNA strand acts as a template to synthesize complementary RNA.
93
Transcription & RNA Processing
- In bacteria, transcribed mRNA is directly functional and translated. - In eukaryotes, genes are split into exons (coding) and introns (non-coding). - Introns are removed by splicing, and exons are joined to produce functional RNA.
94
Genetic Code & Role of tRNA
- mRNA contains triplet codons, each coding for an amino acid. - tRNA is an adapter molecule that binds a specific amino acid at one end. - tRNA pairs with mRNA codons via its anticodon through H-bonding.
95
Translation & Ribosomes
- Translation occurs at ribosomes, where amino acids are joined. - One of the rRNA acts as a ribozyme, catalyzing peptide bond formation. - This process evolved around RNA, suggesting life began with RNA.
96
Gene Expression & Regulation
- Transcription and translation are energy-intensive processes, requiring tight regulation. - In bacteria, genes are arranged into operons. - Lac operon regulates lactose metabolism, controlled by lactose availability in the medium.
97
Human Genome Project – Key Findings
- A mega project aimed at sequencing the entire human genome. - It revealed new information, opening new research avenues. - Less than 2% of the genome codes for proteins. - 99.9% of human DNA is identical, with only 0.1% variation creating uniqueness.
98
DNA Fingerprinting – Principle & Applications
- Works on DNA polymorphism, identifying individual genetic variations. - Forensic Science – Criminal identification from biological samples. - Genetic Biodiversity – Understanding variations within species. - Evolutionary Biology – Studying genetic relationships and ancestry.
