Week 9 - PCR and Molecular Techniques summary

Enzymatic Process:
- Helicase: Unwinds parent DNA at the origin to form the Replication Fork.
- Primase: Synthesizes RNA primers.
- DNA Polymerase III: Moves in a $3'$ to $5'$ direction on the template, synthesizing the new strand in a $5'$ to $3'$ direction.
- Lagging Strand: Synthesized discontinuously as Okazaki fragments.
- Exonuclease (DNA Pol I): Removes RNA primers and replaces them with DNA.
- DNA Ligase: Joins Okazaki fragments together.
Nature: DNA replication is semi-conservative.

Purpose: Separates macromolecules (DNA, RNA, proteins) based on molecular size, charge, and shape.
Physics of Migration: Velocity is defined by $C = \frac{E \times q}{f}$ , where $E$ is the electric field, $q$ is net charge, and $f$ is the frictional coefficient.
Mediums:
- Agarose Gels: Typically $0.5\, \text{--} \,2\%$ concentration; used for DNA and RNA.
- Polyacrylamide Gels (PAGE): Used for proteins and small DNA fragments; involve Acrylamide (a neurotoxin).
SDS-PAGE: Uses Sodium Dodecyl Sulphate (anionic detergent) to give denatured proteins a uniform negative charge for separation by mass.
Visualization:
- Ethidium bromide (EtBr): A potent mutagen and DNA intercalator that fluoresces yellow/orange ( $\sim 590\,nm$ ) under UV light.
- Alternatives: SYBR Safe, GelGreen, EZ-Vision, and Gel Red.

Function: Bacterial endonucleases that cut double-stranded DNA at specific recognition sites ( $4$ , $5$ , $6$ , or $8$ base pairs).
Examples: Eco R1 (from E. coli) and Sma1 (from Serratia marscescens).
Types of Cuts: Can produce "blunt" or "sticky" ends.
Applications: Used in Restriction Fragment Length Polymorphism (RFLP) for paternity testing, forensics, and identifying disease genes.
Pulsed-Field Gel Electrophoresis (PFGE): Used for subtyping bacteria with large chromosomal DNA (> 15 \text{--} 20\,kb).

Origin: Conceived by Kary Mullis in $1983$ at Cetus Corporation.
Reaction Components:
- Template DNA: The target sequence to be amplified.
- Primers: Specific sequences ( $\sim 20\,bp$ ) that bracket the target region.
- Taq Polymerase: A thermostable enzyme isolated from Thermus aquaticus (a hot spring bacterium).
- dNTPs: The building blocks ( $G, C, T, A$ ).
- Buffer: Typically contains $MgCl_2$ , $KCl$ , and $Tris-HCl$ .
Standard Steps:
1. Denaturation ( $95^{\circ}C$ ): Heat separates double-stranded DNA.
2. Annealing ( $50 \text{--} 60^{\circ}C$ ): Primers bind to complementary sequences.
3. Extension ( $72^{\circ}C$ ): Taq polymerase synthesizes new DNA in the $5'$ to $3'$ direction.
Amplification Strategy: Exponential growth represented by $2^n$ , where $n$ is the number of cycles.

Real-Time PCR (qPCR): Quantifies DNA during the reaction using SYBR Green (intercalating dye) or TaqMan probes (utilizing Förster Resonance Energy Transfer or FRET).
Reverse Transcriptase PCR (RT-PCR): Uses mRNA template converted to cDNA for gene expression analysis.
Droplet Digital PCR (ddPCR): Uses water-oil emulsion for absolute quantification and mutation detection.
Clinical/Forensic Uses:
- Detecting pathogens (M. tuberculosis, Chlamydia trachomatis).
- Identifying antibiotic resistance.
- Short Tandem Repeats (STRs): Analysis of non-coding regions for industrial forensics and paternity (e.g., CODIS loci like TH01, TPOX).

First Generation: Sanger sequencing uses fluorescently marked chain-terminating nucleotides to sequence fragments of $100 \text{--} 1000\,bp$ .
Next Generation Sequencing (NGS):
- Ion Torrent: Detects hydrogen ions released during nucleotide incorporation (pH change).
- Illumina: Uses bridge amplification on a glass flow cell and reversible fluorescent blockers.
- Capabilities: Includes Whole-genome sequencing (WGS) and Whole-exome sequencing (WES).
Third Generation: Includes PacBio and Oxford Nanopore for long-read sequencing.