Methods in Molecular Biology: Mutation, DNA Structure, and Molecular Genetic Tools

Mutations and Their Molecular Nature

Classes of Mutagens

Mutagens are agents that cause changes in DNA sequence, leading to mutations. They are classified into three main groups:

• Radiation: Includes UV radiation and X-rays, which can cause DNA damage by inducing breaks or altering bases.

• Chemicals: Such as cigarette smoke, benzene, and hydrogen peroxide, which can modify DNA bases or cause cross-linking.

• Infectious Agents: Viruses (e.g., Human Papillomavirus) and bacteria (e.g., Helicobacter pylori) can integrate into the genome or induce mutations.

Definition and Types of Mutations

A mutation is any change in the DNA sequence. Mutations can occur spontaneously due to errors in biological processes, especially during DNA replication, or be induced by mutagens.

• Spontaneous mutations: Occur even in healthy, uncontaminated cells. The rate depends on the replication system's stringency and repair effectiveness, varying among species and tissue types. Viruses often have higher mutation rates.

Types of Mutations by Molecular Nature

• Nucleotide substitution: One nucleotide is replaced by another.

• Nucleotide insertion: Addition of one or more nucleotides.

• Nucleotide deletion: Removal of one or more nucleotides.

Nucleic Acid Substitutions

• Transitions: Purine to purine (A ↔ G) or pyrimidine to pyrimidine (C ↔ T).

• Transversions: Purine to pyrimidine or vice versa (A/G ↔ C/T).

Mutations Affecting Codons

The genetic code is read in triplets called codons, each coding for one amino acid. Mutations can alter codons, affecting protein synthesis.

• Each single-stranded DNA has three possible reading frames; double-stranded DNA has six.

• Usually, only one reading frame is open (not interrupted by stop codons).

Mutation Type	Effect	Example
Silent	No change in amino acid	Third base change, e.g., GAG (Glu) → GAA (Glu)
Missense	Change in amino acid	Sickle cell: GAG (Glu) → GTG (Val)
Nonsense	Premature stop codon	Cystic fibrosis: CAG (Gln) → TAG (Stop)

Mutations by Nucleotide Insertion/Deletion

• Frameshift: Insertion or deletion not in multiples of three shifts the reading frame, often causing premature stop codons.

• In-frame: Insertion or deletion in multiples of three does not shift the reading frame but may add or remove amino acids.

Mutation	DNA Sequence	Amino Acid Sequence
Normal	CAG CCC ACT	Gln Pro Thr
Insertion (Frameshift)	CAG TTT CCC ACT	Gln Phe Pro Thr
Deletion (Frameshift)	CAG TCC ACT	Gln Ser Thr

Chromosomal Rearrangements

Mutations can also occur at the chromosomal level:

• Inversion: A segment of a chromosome is reversed end to end.

• Translocation: A segment from one chromosome is transferred to another.

DNA Structure and Properties

DNA Double Helix

The DNA double helix consists of two antiparallel strands held together by complementary base pairing and stabilized by base stacking forces.

• Backbone: Hydrophilic (sugar-phosphate), outside.

• Bases: Hydrophobic, inside.

• Hydrogen bonds: Between complementary bases (A-T: 2 bonds, G-C: 3 bonds).

• Base stacking: Major contributor to helix stability.

Denaturation and Renaturation

DNA strands can be separated (denatured) by heating, breaking hydrogen bonds and base stacking. Upon cooling, complementary strands can re-anneal (renature or hybridize).

• Denaturation: Separation of DNA strands by heat or chemicals.

• Renaturation/Hybridization: Reformation of double-stranded DNA when temperature decreases.

Determinants of Melting Temperature ($T_m$)

The melting temperature ($T_m$) is the temperature at which half of the DNA helices are separated.

• DNA length: Longer DNA has higher $T_m$ due to more base stacking and hydrogen bonds.

• GC content: Higher GC content increases $T_m$ because G-C pairs have three hydrogen bonds.

Equation for $T_m$ (approximate):

$T_m = 4^ ext{o}C imes ( ext{number of G/C pairs}) + 2^ ext{o}C imes ( ext{number of A/T pairs})$

Hybridization

Hybridization refers to the process where complementary nucleic acid strands pair to form double-stranded molecules. This is used in various molecular biology techniques:

• DNA-DNA, DNA-RNA, RNA-RNA (inter- and intra-molecular pairing)

• Applications: DNA replication, CRISPR gene editing, RNA interference

Molecular Genetic Tools

Hybridization-Based Techniques

• CRISPR: Uses guide RNA to target specific DNA sequences for gene editing.

• FISH (Fluorescent In Situ Hybridization): Detects and locates specific DNA/RNA sequences in cells using fluorescent probes.

Cloning and PCR

• Cloning: Involves inserting DNA fragments into vectors (plasmids, phage) to create libraries or express genes.

• PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences using cycles of denaturation, annealing, and extension. Requires gene-specific primers.

After 25 cycles, one copy of a gene can be amplified to ~15 million copies.

DNA Polymerase Activity

DNA polymerase synthesizes DNA in the 5' to 3' direction by adding deoxynucleotide triphosphates (dNTPs) to the 3' end, forming phosphodiester bonds.

Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size using an electric current. DNA migrates toward the positive electrode due to its negative charge. Shorter fragments run faster through the agarose gel.

Sanger Sequencing

Sanger sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at specific bases, allowing determination of DNA sequence by fragment length.

Manipulating Genes

• Isolate genes to study function and expression.

• Use reporter constructs (e.g., GFP) to visualize gene expression and protein localization.

• Isolate mutant genes/proteins and make recombinant proteins.

Restriction Enzymes and Cloning Vectors

• Restriction enzymes: Recognize specific DNA sequences (often palindromic) and cleave DNA, enabling recombinant DNA construction.

• Cloning vectors: Plasmids with elements such as origin of replication, antibiotic resistance, metabolic markers, telomeres, and multi-cloning sites (MCS).

Vector Element	Function
Origin of replication (Ori)	Allows vector to replicate in host cells
Antibiotic resistance (ampr)	Selection marker for transformed cells
Metabolic marker	Alternative selection marker
Telomere	Stabilizes linear vectors in eukaryotes
MCS (multi-cloning site)	Region with multiple restriction sites for DNA insertion

Supercoiling and Gel Electrophoresis

Supercoiled DNA migrates faster on agarose gels than linear or relaxed DNA due to its compact structure.

Reporter Vectors and Transgenics

• Reporter vectors (e.g., GFP) are used to study promoter/enhancer activity and protein localization.

• Transgenic animals expressing fluorescent proteins have been commercialized for research and ornamental purposes.

Recombinant Protein Production

• Cloning is essential for producing recombinant proteins (e.g., insulin, human growth hormone, antibodies).

• Proteins can be expressed in bacterial vectors for purification and therapeutic use.

Applications of PCR and Cloning

• Forensics: DNA analysis from small samples (blood, semen, epithelial cells).

• Basic research: Studying gene expression and regulation.

• Genetic disorder identification: PCR-based tests for disease diagnosis.

• Historical mysteries: Ancient DNA analysis (e.g., Neanderthal genes, 1918 flu).

• Genome sequencing: High-throughput sequencing technologies.

Additional info: These notes expand on the original slides by providing definitions, examples, and context for each technique and concept, ensuring a comprehensive understanding suitable for Genetics college students.