BIOL2320_1213
Page 1: Introduction to PCR
Topic: PCR Protocol
Instructor: Kelsie Doering (kelsie.doering@kpu.ca)
Key Techniques: Molecular Techniques, PCR.
Steps mentioned: Get the reagents, Prepare the mix, Set up conditions.
Analyze results via gel electrophoresis; address negative results and the relevance of sketching in science.
Page 2: LessonOutline
Molecular Techniques
Learning Objectives:
Define Single Nucleotide Polymorphism (SNP)and explain its relation to RFLP.
Assess DNA fingerprinting results for identity determination.
Describe the PCR process for amplifying DNA regions.
Design forward and reverse primers.
Explain dideoxynucleotides (ddNTP) and their use in DNA sequencing.
Compare whole genome sequencing (WGS) and whole exome sequencing (WES).
Examine types of gene therapy including target cells and delivery methods.
Problem Set
Chapter references: 5.5, 7.5, 15.3.
Page 3: Genetic Variation Overview
Over 98% of human genome is non-coding.
Many variations in non-coding regions may not impact phenotype.
Utilized historically for gene mapping; known polymorphisms can inform disease gene localization via haplotype analysis.
Example: Individual II4 possesses markers 8-3-8 but shows disorder, suggesting the disease-causing gene lies within that haplotype section.
Page 4: Genetic Markers
DNA Polymorphisms used to narrow down disease genes; categorized as genetic markers.
Types of Genetic Markers:
Single Nucleotide Polymorphisms (SNPs)
Variable Number Tandem Repeats (VNTRs)
Restriction Fragment Length Polymorphisms (RFLPs)
Genetic markers useful in forensics and paternity testing.
Page 5: Single Nucleotide Polymorphisms (SNP)
Definition: Variants in the DNA sequence where one base pair is replaced by another.
Predominantly occur in non-coding regions.
Human genome contains approximately 3.3 million SNPs identified through sequencing.
Page 6: Variable Number Tandem Repeats (VNTRs)
Definition: Repetitive DNA segments found in tandem orientation.
Variation in repeat count between individuals.
Identified through gel electrophoresis.
Page 7: Restriction Fragment Length Polymorphisms (RFLPs)
Definition: Variations in DNA sequences recognized by restriction enzymes.
Produced DNA fragments can be visualized and analyzed via gel electrophoresis.
Page 8: DNA Fingerprinting
Method to identify individuals via unique DNA patterns.
Applications: Paternity testing, forensic analysis, genotyping.
RFLP Fingerprinting: Differentiates DNA fragments based on cutting sites of restriction enzymes.
Tandem Repeat Polymorphism Fingerprinting: Analyzes differences in short tandem repeats among individuals.
Example: Variation in number of repeats indicates identity.
Page 9: Polymerase Chain Reaction (PCR)
Definition: Technique to amplify specific DNA regions in vitro.
Developed by Kary Mullis in 1985; Nobel Prize in Chemistry (1993).
Involves three stages, repeated 25-35 times in a thermal cycler:
Denaturation (96°C): DNA strands separate.
Annealing (50-65°C): Primers attach to the template.
Extension (72°C): DNA polymerase synthesizes new DNA strands.
Page 10: DNA Polymerase in PCR
Essential for synthesizing DNA strands in the 5’ to 3’ direction.
Commonly used enzyme: Taq polymerase from Thermus aquaticus, withstands denaturation temperatures.
Faster alternatives like Phusion polymerase are available.
Page 11: Primers in PCR
Short single strands of DNA designed to hybridize to specific regions of interest.
Two types: Forward and Reverse; both essential for amplification.
Page 12: Properties of Good Primers
Length typically around 20 nucleotides (18-22).
Balanced GC/AT content (50% each).
Include a 3’ G/C clamp to ensure stability during binding.
Should not hybridize to each other.
Page 13: PCR Examples
Example sequence for primers illustrated.
Emphasis on specific primer design for amplification success.
Page 14: Conducting PCR
Components needed in PCR: Template DNA, dNTPs, Forward/Reverse primers, Buffer, Taq polymerase.
Process involves running the reaction in a thermal cycler and analyzing products via gel electrophoresis.
Page 15: Gel Electrophoresis Overview
Technique for visualizing PCR or cloning outcomes.
DNA fragments are separated by size through a gel matrix under an electric current.
Agarose gel is the medium; size determines movement through the gel.
Page 16: Loading DNA into Gel
DNA is loaded into gel wells; movement towards positive electrode due to DNA's negative charge.
Page 17: Visualizing DNA Fragments
Staining methods used to visualize separated DNA bands; common stains: Ethidium bromide (carcinogenic) and SYBR Safe.
Importance of including a DNA ladder for size reference.
Page 18: PCR Genotype Analysis Question
PCR used to determine genotypes for gene A.
Discussion and interpretation of results regarding allele presence in individuals.
Page 19: DNA Sequencing Overview
Process of determining nucleotide sequences in DNA.
Requires DNA fragmentation, sequencing of pieces, and genome reassembly.
Historical context of the Human Genome Project.
Page 20: Sanger Sequencing Technique
Developed in 1977 by Fred Sanger; suitable for sequences around 900bp.
Requires similar components to PCR with the addition of ddNTPs.
Page 21: Dideoxynucleotides (ddNTPs)
Definition: Nucleotides preventing further DNA strand extension, leading to chain termination.
Page 22-23: Sanger Sequencing Process
Older Sanger method involved multiple tubes for sequencing; each with one ddNTP variant.
Explanation of the process from primer annealing to band separation.
Page 24: Sequence Determination Example
Given PCR results, deducing the template DNA sequence from the identified new strand.
Page 25: Advanced Sanger Sequencing
Modern Sanger sequencing involves simultaneous reactions with labeled ddNTPs.
Capillary gel electrophoresis separates fragments for automated sequence reading.
Page 26: Introduction to Next Generation Sequencing (NGS)
Common features: Parallel processing, micro scale, fast results, low cost, shorter reads.
Page 27: Whole Genome vs Whole Exome Sequencing
WGS sequences entire genome; WES targets only exons.
WGS detects coding and noncoding variants; WES focuses only on exons.
Page 28: Gene Therapy Introduction
Definition: Using genes to treat or prevent diseases; can involve adding or fixing genes.
Page 29: Types of Gene Therapy
Somatic Gene Therapy targets body cells (not inheritable)
Germline Gene Therapy targets germ cells (inheritable changes).
Page 30-31: Methods of Gene Delivery
In vivo: Direct delivery to cells within the body.
Ex vivo: Cells modified outside the body and transplanted back.
Page 32: Gene Replacement Therapy
Delivers wild-type genes to defective cells to restore function;
Utilizes cloning techniques for gene production.
Page 33: Glybera Case Study
Gene therapy for Lipoprotein lipase deficiency; costly single-treatment approach.
Page 34: Gene Editing Overview
Techniques: ZFNs, TALENs, CRISPR; CRISPR considered the most advanced and efficient.
Page 35: CRISPR/Cas9 Mechanism
CRISPR as a bacterial immune system; modified for genome editing applications.
Page 36: CRISPR Mechanics
Double strand break repairs: Non-Homologous End Joining and Homology Directed Repair.
Page 37: CRISPR Components
Key components: Guide RNA for target genes and Cas9 for cutting DNA strands.
Page 38: Current CRISPR Applications
FDA-approved therapies and ethical considerations surrounding gene editing in embryos.
Page 39: Stem Cells in Gene Therapy
Extend treatment possibilities beyond typical in vivo methods; importance of targeting stem cells.
Page 40: Stem Cell Potency
Classification of stem cell potency and relevance in genetic therapies.
Page 41: Induced Pluripotent Stem Cells (iPSC)
Reprogramming adult cells to pluripotency; implications for overcoming immune response issues.
Page 42: Steps for iPSC and CRISPR Integration
Detailed steps for using iPSC in conjunction with CRISPR for targeted therapies.