SCH1111 Fundamental Biomedical Techniques: PCR & Other Molecular Techniques

Topic = PCR and other molecular techniques

PCR and Other Molecular Techniques

Conceptual Overview:
DNA replication is described as a semi-conservative process, meaning each new DNA molecule consists of one parental strand and one newly synthesized strand.
The overall direction of replication proceeds from the origin of replication, creating a replication fork.

Enzymatic Breakdown and Mechanism:

Helicase: Unwinds a portion of the parent DNA at the origin, forming the replication fork.
Single-strand binding protein: Stabilizes the unwound parental DNA strands to prevent them from re-annealing.
Primase: Designs and synthesizes RNA primers that provide a starting point for DNA synthesis.
DNA Polymerase III (DNA pol III): Binds to the template strands and moves in a $3'$ to $5'$ direction. It reads the sequence and replicates the new strand in a $5'$ to $3'$ direction.
Leading Strand: Synthesized continuously in the same direction as the replication fork movement ( $5'$ to $3'$ ).
Lagging Strand: Synthesized discontinuously in the direction opposite to the replication fork. This requires multiple primers.
Okazaki Fragments: The short segments of DNA synthesized on the lagging strand.
DNA Polymerase I (DNA pol I): An exonuclease enzyme that removes RNA primers and replaces them with DNA nucleotides.
DNA Ligase: Joins (ligates) Okazaki fragments together to create a continuous DNA strand.

DNA Transcription and Translation

The Central Dogma Process:
Transcription: Occurs in the nucleus where DNA (the Gene) is transcribed into pre-mRNA.
mRNA Processing: Involved in converting pre-mRNA into mature mRNA via several steps:
Capping: Addition of a $5'$ cap.
Splicing: Excision of Introns (non-coding regions) and splicing together of Exons (coding regions).
Polyadenylation: Addition of a Poly(A) tail at the $3'$ end.
Export: The processed mRNA is exported from the nucleus into the cytoplasm.
Translation: Occurs at the ribosome in the cytosol, where the mRNA sequence is translated into a growing protein chain, which then folds into a functional protein.
The Human Protein Atlas: An open-access resource for human proteins used to search for specific genes or proteins (e.g., $ACE2$ , $GFAP$ , $EGFR$ , $TP53$ , $NEFL$ , or $VEGFB$ )

Gel Electrophoresis: Methodology and Physics

Definition: A method for the separation and analysis of macromolecules (DNA, RNA, and proteins) and their fragments based on molecular size, charge, and shape.
Physics of Migration:
The velocity of migration is determined by the formula: $C = \frac{E \times q}{f}$
Where:
$C$ = velocity of migration
$E$ = electric field in $\text{volt/cm}$
$q$ = net electric charge on the molecule
$f$ = frictional coefficient

Principles of Movement:
Molecules move toward the positive pole (Anode) when an electrical potential is applied.
Size: Smaller molecules travel faster and further down the gel than large molecules.
Shape/Conformation: Open DNA conformations travel slower than supercoiled or globular conformations.
Buffer pH: Can affect the charge of the molecule and its subsequent migration.

Types of Gel Matrices

Agarose Gels:
Used for the separation of DNA and RNA.
Agarose is a linear carbohydrate (polysaccharide) extracted from seaweed.
In solution, it shifts from random coils to a structure where chains are bundled into double helices.
Concentration: Typically used at $0.5\% - 2.0\%$ . Higher percentages create smaller pores, which are better for separating small molecules.

Polyacrylamide Gels (PAGE):
Commonly used for protein separation but can also separate DNA.
Formed from the polymerization of acrylamide and $N,N’\text{-methylenebisacrylamide}$ .
Safety Warning: Acrylamide is a potent neurotoxin and must be handled with extreme care.
SDS-PAGE: Uses Sodium Dodecyl Sulphate (an anionic detergent) to bind denatured proteins, giving them a uniform high-density negative charge so they migrate toward the Anode based on size alone.
Reducing Agents: $\beta\text{-mercaptoethanol}$ is used to reduce disulfide bonds in proteins.

Agarose Electrophoresis Procedures and Visualization

Laboratory Steps:
1. Prepare agarose gel in a buffer such as TAE (Tris-acetate-EDTA) or TBE (Tris-borate-EDTA).
2. Melt agarose, pour into a gel chamber with a 'comb' inserted, and allow to set ( $10\text{-}20\,\text{min}$ ).
3. Place gel in the tank and cover with the same buffer used for making the gel.
4. Mix DNA samples with Loading Dye (containing a color agent and glycerol, sucrose, or ficoll to ensure samples sink into wells).
5. Load a DNA ladder in the first well as a reference.
6. Load samples and run at approximately $90\text{-}100\,\text{V}$ for $30\text{-}60\,\text{min}$ .
7. Visualize under UV light using a gel doc scanner.

Visualization Agents:
Ethidium Bromide (EtBr): A DNA intercalator that inserts into spaces between base pairs. It absorbs UV light ( $300\text{ nm}$ and $360\text{ nm}$ ) and emits visible yellow/orange light at approximately $590\text{ nm}$ .
Safety Warning: Ethidium bromide is a potent mutagen; solutions must be handled with caution and decontaminated before disposal.
Alternatives: SYBR Safe, GelGreen, EZ-Vision, Gel Red.

Applications of Gel Electrophoresis

General Uses:
To examine isolated DNA.
To analyze DNA fragments cut with restriction enzymes.
To examine DNA amplified via PCR.
Pulsed-Field Gel Electrophoresis (PFGE):
The "gold standard" for bacterial subtyping (established in 1984).
Used for bacteria with large chromosomal DNA.
Handles molecules larger than $15\text{-}20\,\text{kb}$ , which would otherwise move together in a size-independent manner. It uses an alternating voltage gradient to improve resolution.

Restriction Enzymes and RFLP

Restriction Endonucleases: Bacterial enzymes that cut double-stranded DNA at specific nucleotide sequences called recognition sites (typically $4, 5, 6,\text{ or } 8$ base pairs long).
Function: Naturally protects bacteria from foreign DNA (viruses). Over 600 enzymes exist for over 200 recognition sites.
Cut Types: Can produce "blunt ends" or "sticky ends."
Eco R1: Isolated from $E. coli$ .
Sma1: Isolated from $Serratia marscescens$ .
Restriction Fragment Length Polymorphism (RFLP):
Identifies organisms by analyzing patterns derived from DNA cleavage.
Used in paternity testing, criminal cases, and locating disease genes.

The Polymerase Chain Reaction (PCR)

Conceptual Overview: A technique to amplify a small amount of template DNA (or RNA) into large quantities within a few hours. This makes scarce genetic material abundant for experimentation.

History:
Conceived by Kary Mullis in 1983 at Cetus Corporation.
Awards: Nobel Prize for Chemistry (1993), Thomas A. Edison Award (1993), Californian Scientist of the Year (1992).

PCR Amplification Mathematics:
Exponential amplification follows the formula: $\text{Copies} = 2^n$
Where $n$ is the number of cycles.
Cycle 1 = $2^1 = 2$ copies.
Cycle 2 = $2^2 = 4$ copies.
Cycle 3 = $2^3 = 8$ copies.
Cycle 4 = $2^4 = 16$ copies.

Technical Components and Steps of PCR

Reaction Reagents:
1. Template DNA: Double-stranded DNA ( $1\text{-}1000\,\text{ng}$ ).
2. Primers: Forward and Reverse ( $10\text{-}20\,\text{pmol}$ ). Usually around $20\,\text{bp}$ long.
3. Taq Polymerase: Thermostable enzyme ( $2^1\,\text{units}$ ) isolated from $Thermus aquaticus$ , a bacterium from hot springs (Yellowstone Park).
4. dNTPs: Building blocks (G, C, T, A) at $50\,\text{mM}$ each.
5. PCR Buffer: $10\,\text{mM Tris-HCl}$ (pH 9.0), $50\,\text{mM KCl}$ , and $0.5\text{-}3.0\,\text{mM MgCl}_2$ .
6. Total Volume: $10\text{-}50\,\mu\text{l}$ .

The Three PCR Steps:
A) Denaturation ( $95^\circ\text{C}$ ): Heat unwinds the double-stranded DNA into single strands.
B) Annealing ( $50\text{-}60^\circ\text{C}$ ): Mixture is cooled to allow primers to base-pair (anneal) to the template at specific sequences.
C) Extension ( $72^\circ\text{C}$ ): Taq polymerase synthesizes complementary DNA in the $5'$ to $3'$ direction by adding dNTPs.

Equipment: A thermal cycler (PCR machine) consisting of a computer-programmed thermal block.
Analyzing Results:
Run on agarose gel with Ethidium Bromide.
Success: A single clear band.
Failure: Multiple bands (non-specific binding) or a smear (degradation or poor optimization).

PCR Variations

Real-time PCR (qPCR):
Allows quantifiable detection of PCR product during the reaction (unlike endpoint PCR).
Detection Methods:
1. SYBR Green: Intercalating dye that emits fluorescence when bound to newly synthesized DNA.
2. TaqMan Probes: Uses Förster Resonance Energy Transfer (FRET). A probe with a reporter and a quencher is cleaved by DNA polymerase during extension, allowing the reporter to emit a signal.

Reverse Transcriptase PCR (RT-PCR) and qRT-PCR:
Uses mRNA as a template.
Reverse Transcriptase converts mRNA into complementary DNA (cDNA) using random hexamers or oligo-dT primers.
Used for gene expression analysis.

Droplet Digital PCR (ddPCR):
Based on water-oil emulsion droplet technology.
Useful for mutation detection, copy number variation, and accurate gene expression.
PCR-RFLP: Combines PCR with restriction enzyme digestion for pattern analysis.

Applications of PCR

Diagnostics:
Identifying genetic conditions (Muscular Dystrophy, Cystic Fibrosis, Breast Cancer).
Detecting pathogens ( $M. tuberculosis$ , $Mycoplasma pneumoniae$ , $Bordetella pertusis$ , $Neisseria gonorrhea$ , $Chlamydia trachomatis$ ).
Identifying antibiotic resistance.

Forensics and Paternity:
Short Tandem Repeats (STRs): 2–7 bp sequences repeating consecutively in non-coding regions.
Y-STRs: Paternal lineage markers (DYS391, DYS385, DYS438).
CODIS loci: TH01, TPOX, vWA, CSF1PO, FGA.
Research: Mutation analysis, cloning, and evolutionary relationship studies.

DNA Sequencing Technologies

First Generation: Sanger Sequencing:
Uses chain termination with fluorescent nucleotides to create fragments of different lengths ( $100\text{-}1000\,\text{bp}$ ).

Second Generation: Next Generation Sequencing (NGS):
Shotgun Sequencing: Randomly fragments long DNA for computer assembly.
Ion Torrent (Semiconductor Sequencing): Uses a chip with 5-micron beads. When a nucleotide is incorporated, a hydrogen ion is released, lowering the pH, which is detected by the chip.
Illumina (Bridge Amplification): DNA is amplified on a glass flow cell containing nanowells. Fluorescent blockers allow the addition of only one nucleotide at a time for recording before being washed away.
Types of NGS: Whole-genome (WGS), Whole-exome (WES), Whole-transcriptome, and Targeted sequencing.

Third Generation:
Long-read sequencing (PacBio, Oxford Nanopore, Illumina Novaseq Series).

History of Genetics and the Human Genome Project (HGP)

Landmarks:
1865: Mendel discovers principles of genetics.
1944: Avery, MacLeod, and McCarty demonstrate DNA is hereditary material.
1953: Watson and Crick describe the double-helical structure of DNA.
1977: Sanger and others develop DNA-sequencing methods.
1984: PCR invented by Kary Mullis.
1990: HGP launched in the US.
2000: Draft version of the human genome completed.
2003: HGP ends with the finished version of the human genome sequence.

Review Questions

Q1: What is a PCR and what can it be used for?
Q2: List the components of a PCR reaction and explain their function.
Q3: List and explain the steps and cycles in the PCR reaction.
Q4: What is agarose gel electrophoresis and what is it used for?
Q5: Describe the steps involved in making an agarose gel for separation of DNA fragments between $1\,\text{kb}$ and $500\,\text{bp}$ .
Q6: How can you use PCR to test for a genetic disease caused by a particular mutation in DNA? Explain the results of such a test.
Q7: Provide examples of PCR applications for bacterial analysis.