Definitions [1]
Definition: DNA Fingerprinting
The technique of identifying an individual by analyzing the unique DNA sequence present in each person, similar to fingerprints, is called DNA fingerprinting.
Key Points
Key Points: Introduction of Molecular Basis of Inheritance
- Nucleic acids are polymers of nucleotides and serve as the building blocks of genetic material.
- Two types of nucleic acids found in living systems are DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid).
- DNA acts as the genetic material in most organisms; it was identified as the genetic material over a century after Mendel's era.
- RNA acts as genetic material in some viruses; in other organisms, it primarily functions as a messenger (mRNA) and also acts as an adapter, structural and catalytic molecule.
- Human genome sequencing has opened a new era of genomics, building on the understanding of DNA as genetic material.
Key Points: The Experimental Proof
- In 1958, Meselson and Stahl proved that DNA replicates semi-conservatively in E. coli.
- E. coli was first grown in heavy nitrogen (¹⁵N) medium, making all DNA heavy (¹⁵N-¹⁵N); cells were then transferred to normal nitrogen (¹⁴N) medium.
- DNA samples were separated using CsCl (caesium chloride) density gradient centrifugation to distinguish heavy, light and hybrid DNA.
- After one generation in ¹⁴N medium, DNA showed hybrid/intermediate density (¹⁵N-¹⁴N); after two generations, DNA was half hybrid + half light (¹⁴N-¹⁴N).
- In 1958, Taylor and colleagues also proved semi-conservative replication in Vicia faba (faba beans) using radioactive thymidine.
Key Points: The Machinery and the Enzymes
- Main enzyme = DNA-dependent DNA polymerase; polymerises at ~2000 bp/sec; mistakes cause mutations.
- Deoxyribonucleoside triphosphates act as substrates and provide energy for polymerisation.
- Replication occurs at the replication fork; only in the 5' to 3' direction; one strand = continuous, the other = discontinuous; fragments are joined by DNA ligase.
- Replication starts at a fixed point called the origin of replication; DNA polymerase cannot initiate randomly.
- In eukaryotes, replication occurs in the S-phase; failure of cell division after replication causes polyploidy.
Key Points: The Lac Operon
- The lac operon is an inducible operon in E. coli, proposed by Jacob and Monod (1961), that controls the metabolism of lactose.
- It consists of a regulator gene (i), promoter (P), operator (O), and three structural genes - lac z, lac y, lac a - coding for β-galactosidase, permease, and transacetylase respectively.
- When lactose is absent: the regulator gene produces an active repressor that binds the operator and blocks RNA polymerase, so the operon remains switched OFF.
- When lactose is present: lactose is converted into allolactose (the inducer), which binds the repressor and inactivates it, leaving the operator free.
- RNA polymerase then transcribes the structural genes into a single polycistronic mRNA, producing the enzymes that break down lactose - the operon is now switched ON.
Key Points: Human Genome Project
- The Human Genome Project (HGP) was an international mega-project launched in 1990 and completed in 2003, coordinated mainly by the U.S. DOE and NIH.
- Its aim was to identify all human genes and sequence the entire human genome of about 3 billion base pairs.
- The main goals were to identify genes, sequence the genome, store the data, develop analysis tools, transfer technologies, and address ethical issues.
- Methodology: DNA was isolated, fragmented, cloned into vectors like BACs and YACs, sequenced by automated methods, and assembled using computers.
- Salient features: the genome has ~3 billion base pairs and 20,000–25,000 genes; less than 2% codes for proteins, and humans are 99.9% identical.
- Most genetic variation between individuals is due to SNPs (single-nucleotide polymorphisms).
- Applications: disease gene mapping, early diagnosis, personalised medicine, evolutionary studies, and advances in biotechnology.
- ELSI (Ethical, Legal, Social Issues): genome data must be kept confidential to prevent misuse and discrimination.
Key Points: DNA Fingerprinting
- DNA fingerprinting is a technique used to identify an individual by analysing the unique DNA pattern present in every person (except identical twins).
- It is based on satellite DNA, especially VNTRs (Variable Number Tandem Repeats) - short sequences repeated in tandem, whose number varies among individuals and creates DNA polymorphism.
- Principle: the differences in VNTR repeat number produce DNA fragments of different lengths, which appear as a unique banding pattern.
- Steps: DNA isolation → PCR amplification → restriction digestion → gel electrophoresis → Southern blotting → probe hybridisation → autoradiography → comparison of band patterns.
- Applications: forensic identification, paternity/maternity testing, pedigree studies, medical research, conservation biology, and evolutionary/anthropological studies.
