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

1. 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.

2. 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:

  1. Single Nucleotide Polymorphisms (SNPs)

  2. Variable Number Tandem Repeats (VNTRs)

  3. 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:

  1. Denaturation (96°C): DNA strands separate.

  2. Annealing (50-65°C): Primers attach to the template.

  3. 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.