DNA Sequencing

Page 1: Title and Introduction

DNA SequencingPresented by: Dr. Louise RobinsonContact: L.Robinson@derby.ac.uk

Page 2: Aims of the Presentation

  • Understand Sanger sequencing, the first-generation process of DNA sequencing developed in 1977, which remains in use today.

  • Gain awareness of next-generation sequencing (NGS) and its applications.

  • Engage with supplementary videos and simulations available on Blackboard to aid comprehension.

Page 3: Understanding DNA Sequencing

Definition and Importance

  • DNA sequencing is the process of determining the order of nucleotides (the building blocks of DNA) in a specific DNA fragment.

  • Sequence analysis plays a significant role across various scientific disciplines, including:

    • Human and non-human identification.

    • Disease analysis.

    • Evolutionary studies.

    • Investigation of genetic disorders and ancestry.

Page 4: Historical Development of DNA Sequencing

Key Milestones in DNA Sequencing

  1. 1965: Robert Holley and colleagues achieved the first complete nucleic acid sequence of alanine tRNA from yeast (Saccharomyces cerevisiae).

  2. 1972: Walter Fiers’ lab produced the first complete protein-coding gene sequence (coat protein of bacteriophage MS2).

  3. 1970: Initial attempts with type II restriction enzymes yielded limited results.

  4. 1975-1977: Allan Maxam and Walter Gilbert developed a chemical modification-based method for DNA sequencing.

Page 5: Sanger Sequencing

Introduction to First-Generation Sequencing

  • Established in 1977, Sanger sequencing employs sequence-specific termination during in-vitro DNA synthesis using modified nucleotide bases.

  • Up until the mid-2000s, the chain termination method was the predominant technique for DNA sequencing.

  • Frederick Sanger’s research group at Cambridge developed this approach utilizing di-deoxy chain termination sequencing. The results were initially read manually using a gel and radioactive tagging.

Page 6: Methodology of Sanger Sequencing

Steps Involved

  • Template DNA: The DNA extension begins at a designated site on the template.

  • Primer: A complementary short oligonucleotide primer is used, with only one primer in Sanger sequencing.

  • DNA Polymerase: The oligonucleotide primer is elongated using DNA polymerase along with all four deoxynucleotide triphosphates (dNTPs).

Page 7: Di-deoxy Nucleotides

Characteristics of the Terminators

  • Di-deoxy nucleotides (ddNTPs) lack a 3’ hydroxyl group, preventing subsequent nucleotide addition.

  • These chain-terminating nucleotides are utilized at a low concentration compared to dNTPs to allow for effective sequence determination.

  • Types of ddNTPs include: ddGTP, ddATP, ddTTP, and ddCTP.

Page 8: DNA Structure Overview

Comparison of DNA and RNA

  • DNA (Deoxyribonucleic Acid): - Double-stranded with deoxyribose sugars and specific base pairs (A-T, C-G).

  • RNA (Ribonucleic Acid): - Single-stranded with ribose sugars, containing uracil instead of thymine.

  • Directionality is a critical aspect: DNA is anti-parallel with a 5' to 3' orientation.

Page 9: Sequencing Process in Sanger

Polymerase Activity

  • DNA polymerase adds deoxynucleotides directed by the matching base on the template strand. This process is termed template-directed DNA sequencing.

  • The available bases for polymerase include normal deoxynucleotides and terminating di-deoxynucleotides. When a ddNTP is incorporated, chain elongation ceases, leading to varied lengths of strands.

Page 10: Transitioning to Single-Stranded DNA

Process Details

  • Due to the vast number of strands being replicated, ddNTP incorporation results in a mixture of fragments of differing lengths, allowing for sequence determination based on strand size.

  • The subsequent step involves converting double-stranded DNA into single-stranded DNA for effective sequencing.

Page 11: Sequence Analysis Techniques

Fragment Analysis

  • Sequencing reactions and fragments are loaded onto gels, where they are separated by electrophoresis.

  • Initially utilized slab electrophoresis with a polyacrylamide gel matrix for high sensitivity; currently, capillary electrophoresis using narrow glass tubes for enhanced efficiency.

Page 12: Historical Analysis of Sequencing

Evolution of Techniques

  • Early methods using x-ray film and manual reading of bands from chain termination events have evolved into automated processes with tagged bases. Capillary electrophoresis of Sanger sequencing was established in 1995.

Page 13: Capillary Electrophoresis Method

Modern Approaches

  • Each strand undergoes sequencing numerous times to enhance fluorescence intensity, ensuring accurate reading as they approach and cross a sensor.

Page 14: Limitations of Sanger Sequencing

Challenges Faced

  • Despite providing excellent sequence reads, Sanger sequencing is relatively slow and inefficient for extensive genomic sequencing, such as the Human Genome Project, which cost $3 billion and consumed 13 years. This necessitated the advent of next-generation sequencing (NGS).

Page 15: Introduction to Next-Generation Sequencing (NGS)

Overview

  • Around 2005, NGS arose, enabling small fragments to be sequenced and subsequently pieced together using computational methods, targeting lengths of 100-300 base pairs. Different NGS platforms include SOLID v4, HiSeq2000, GAllx, and GS-FLX.

Page 16: NGS Techniques Overview

Steps in Next-Generation Sequencing

  1. Library Preparation: Preparing the DNA library for sequencing.

  2. Clonal Amplification: Amplifying the library to create sufficient DNA.

  3. Cyclic Array Sequencing: This includes processes like emulsion PCR, pyrosequencing, and sequencing-by-synthesis. Techniques also encompass DNA ligase for sequencing via ligation.

Page 17: Cost and Time Revolution

Advancements in Sequencing

  • The cost of genome sequencing has dramatically declined from billions to approximately $1000, with the timeline reducing from 13 years to mere hours.

Page 18: Practical Applications of Whole-Genome Sequencing

Tailored Cancer Care

  • The NHS is leveraging whole-gene screening to customize cancer treatments based on genetic profiling from both healthy inherited genomes and corrupted cancer genomes.

  • Recent studies involving 13,000 cancer patients emphasize the potential of incorporating genetic data into standard treatment protocols. Genetic variations can inform drug responses, such as the BRCA1 mutation’s impact on breast and ovarian cancer susceptibility.

  • This developments highlight the importance of long-term clinical data fused with genomic information to enhance treatment outcomes and patient management.

Page 19: Innovations in Third Generation Sequencing

MinION Technology

  • Advances in sequencing technologies include third-generation sequencing through devices like MinION, facilitating real-time analysis.

Page 20: Research Case Study

Biodiversity Sampling via Nanopore Technology

  • Researchers are using dung beetles and their stomach contents for DNA sequencing as a groundbreaking method to analyze biodiversity. The Nanopore minION device allows real-time data collection and identification of species from DNA samples efficiently.