Unit–3: DNA Sequencing, PCR and Molecular Analysis
(Detailed Notes)
Primary References: T. A. Brown – Gene Cloning and DNA Analysis; Smita Rastogi & Neelam Pathak – Genetic
Engineering
Prepared for: [Link]. Microbiology (University Examination + CSIR/UGC NET)
1. DNA Sequencing: Introduction
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It is
essential for genome analysis, gene identification, mutation detection and evolutionary studies.
2. Maxam–Gilbert Sequencing Method
Also known as the chemical degradation method, Maxam–Gilbert sequencing is based on base-specific chemical
cleavage of DNA.
Principle
• End labeling of DNA with radioactive phosphate
• Chemical modification of specific bases
• Cleavage at modified bases
• Fragment separation by polyacrylamide gel electrophoresis
Advantages and Limitations
• High specificity
• Use of toxic chemicals
• Labor-intensive and not suitable for automation
3. Sanger Sequencing Method
Also called the dideoxy chain termination method, Sanger sequencing is the most widely used classical DNA
sequencing technique.
Principle
• Use of dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP)
• Termination of DNA synthesis
• Generation of fragments of varying length
• Detection by autoradiography or fluorescence
Advantages
• High accuracy
• Automation possible
• Widely used in small-scale sequencing
4. Shotgun Sequencing
Shotgun sequencing involves random fragmentation of DNA followed by sequencing of individual fragments and
computational assembly.
• Random DNA fragmentation
• Cloning or direct sequencing
• Sequence assembly using overlaps
• Used in whole genome sequencing
5. Next Generation Sequencing (NGS)
Next generation sequencing technologies allow massively parallel sequencing of millions of DNA fragments
simultaneously.
General Features
• High throughput
• Reduced cost per base
• Short read lengths
• Massive data output
Applications
• Whole genome sequencing
• Transcriptomics
• Metagenomics
• Cancer genomics
6. DNA Mapping and Fingerprinting
DNA Mapping
• Physical mapping – restriction maps, contig maps
• Genetic mapping – linkage analysis
DNA Fingerprinting
DNA fingerprinting is based on detection of polymorphic DNA regions.
• RFLP – restriction fragment length polymorphism
• PCR-based methods – RAPD, AFLP, STRs
• Applications in forensics and population studies
7. Site-Directed Mutagenesis
Site-directed mutagenesis is used to introduce specific, deliberate changes in a DNA sequence.
• Oligonucleotide-directed mutagenesis
• PCR-based mutagenesis
• Applications in protein engineering and functional analysis
8. Polymerase Chain Reaction (PCR)
PCR is an in vitro technique for exponential amplification of a specific DNA sequence.
Basic Steps
• Denaturation
• Annealing
• Extension
Types of PCR
• Reverse transcription PCR (RT-PCR)
• Real-time PCR (qPCR)
• Multiplex PCR
• Nested PCR
9. Construction of Genomic and cDNA Libraries
Genomic Library
• Represents entire genome
• Cloned in plasmids, cosmids, BACs or YACs
• Used for genome mapping
cDNA Library
• Prepared from mRNA using reverse transcriptase
• Represents expressed genes
• Used for expression studies
10. Exam-Oriented Notes (University + NET)
• Differentiate Sanger vs Maxam–Gilbert clearly
• Write steps of PCR with temperature values
• Compare genomic vs cDNA libraries
• NET focus: PCR variants and NGS features