Lecture 16 DNA Sequencing (2).pptx

Next Section: DNA Sequencing RDNA202 Cassie [email protected] Lectures Lecture 16: DNA Sequencing Theory & Sanger Sequencing Lecture 17 & 18: DNA Sequencing Theory & NGS  Sanger Sequencin g RDNA202 Cassie [email protected] https://www.cbehx...

Next Section: DNA Sequencing RDNA202 Cassie [email protected] Lectures Lecture 16: DNA Sequencing Theory & Sanger Sequencing Lecture 17 & 18: DNA Sequencing Theory & NGS  Sanger Sequencin g RDNA202 Cassie [email protected] https://www.cbehx.co.uk/product/next-generation-sequencing-ngs/ DNA Sequencing The process of determining the order of bases in a length of DNA Has helped advance our understanding of genetics. Applications of DNA Sequencing Tackling Human Disease Identification of mutations associated with diseases – e.g. cancer Identifying variations and polymorphisms In the human genome Provides information about Single Nucleotide Polymorphisms (SNPs) Studying evolution: How genes and genomes evolve Sanger Sequencing method Also known as “chain termination method” Classified as a First Generation Sequencing method Used to determine the nucleotide sequence of DNA Developed in 1977 by two time Nobel Laureate – Frederick Sanger and his colleagues Father of genomics: 13 August 1918 – 19 November 2013 Fred and colleagues – first scientists to sequence whole genome Genome was just over 5000 bases – from virus called phiX174 – infects bacteria Sanger Sequencing method Uses high fidelity DNA-dependent polymerase Create a complementary copy to a single stranded DNA template Single primer – complementary to template Initiates DNA synthesis from 3’ end. Sanger Sequencing – Chain Termination Method Sanger Sequencing - ddNTPs Resemble DNA monomers Incorporated into growing chain Differ from dNTPs in two ways: 1. Lack 3’ hydroxyl group required for further DNA extension Leads to chain termination once incorporated 2. Each ddNTP has fluorescent dye attached Allows for automatic detection Nucleotides: Deoxytriphosphate Vs Dideoxytriphosphate  Components Required for Sanger Sequencing Note: Only ONE Primer is used Sanger Sequencing Method – Step 1 1. The DNA to be sequenced must first be amplified by PCR Then DNA double helix is denatured with heat Separates the two strands These will be the templates for DNA synthesis  Sanger Sequencing Method – Step 2 2. Generation of n DNA fragments of varying lengths Each terminated with a fluorescently labelled nucleotide Where n = the number of nucleotide bases in the target DNA sequence This is done by combining: DNA primer Nucleotides – deoxynucleotide triphosphates (dNTPs =dATP, dTTP, dCTP, dGTP) Labelled Dideoxynucleotides (ddNTPs = ddATP, ddTTP, ddCTP, ddGTP)  Sanger Sequencing Method – Step 2 continued 2. Labelled Dideoxynucleotides (ddNTPs = ddATP, ddTTP, ddCTP, ddGTP) No nucleotide can be added to the chain once a ddNTP has been added Each fragment will end with a labelled nucleotide Therefore – much smaller amount of ddNTPs are used compared to the normal dNTPs  Sanger Sequencing Method: Chain Termination GROWING DNA CHAIN TERMINATES DUE TO INCORPORATION OF A DDNTP. Sanger Sequencing Method – Step 3 3. Capillary electrophoresis Separate amplicons of various lengths These amplicons – terminated with ddNTP at 3’ end Principles of electrophoresis: Shorter fragments move faster than longer fragments Results in DNA being analysed from shortest to longest sequence Sanger Sequencing Method – Capillary electrophoresis Shorter fragments move faster than longer fragments Sanger Sequencing Example: Reading Results  Sanger Sequencing Method: The process The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with a mixture of all four normal (deoxy) nucleotides in ample quantities dATP dGTP dCTP dTTP a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color: ddATP ddGTP ddCTP ddTTP, and DNA polymerase I Sanger Sequencing Method – Step 4 4. In the final step: Laser excites label on nucleotide at the end of each sequence Each base is tagged with a different label Light emitted by each excited nucleotide – tied to correct base Laser generates a chromatogram Shows fluorescent peak of each nucleotide Nucleotides are in correct order because of electrophoresis. Sanger Sequencing Method – Step 4 Sanger Sequencing Method – Step 4 Sanger Sequencing Overview  DNA Sequencing: Next Generation Sequencing (NGS) RDNA202 Cassie [email protected]