dnasequencing1.pptx

DNA SEQUENCING 02/24/2025 1  DNA SEQUENCING Determining the order of bases in a section of DNA 02/24/2025 2  PURPOSE Deciphering “code of life” Detecting mutations Typing mic...

DNA SEQUENCING 02/24/2025 1  DNA SEQUENCING Determining the order of bases in a section of DNA 02/24/2025 2  PURPOSE Deciphering “code of life” Detecting mutations Typing microorganisms Identifying human haplotypes Designating polymorphisms 02/24/2025 3  DNA SEQUENCING METHODS Historically there are two main methods of DNA sequencing: 1. Maxam and Gilbert method 2. Sanger method 02/24/2025 4  Maxam and Gilbert Method Walter Gilbert Allan Maxam 1976–1977 Principle Chemical degradation of purines and pyrimidines  Maxam and Gilbert Method  The single stranded DNA fragment to be sequenced is treated with alkaline phosphatase to remove the 5’phosphate  P-labeled ATP added to the 5’terminal in the presence of polynucleotide kinase,  The labeled DNA fragment is then divided into 4 aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine  The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA at the residue that has been modified  Maxam-Gilbert sequencing - modifications Maxam-Gilbert sequencing Pyrosequencing 9 Pyrosequencing 10  Disadvantages  Requires lots of purified DNA, and many intermediate purification steps  Relatively short readings  Automation not available (sequencers)  Remaining use for ‘footprinting’ (partial protection against DNA modification  Sanger Method Most common approach used to sequencing DNA. Also termed as chain termination or dideoxy method Invented by Frederick Sanger – 1977 Nobel prize – 1958 & 1980  SANGER METHOD This method uses dideoxynucleotide triphosphates (ddNTPs) chain terminators : “which has an H on the 3’ carbon of the ribose sugar instead of the normal OH found in dNTPs”. if a ddNTP is added instead of the normal dNTP, the synthesis stops at that point because the 3’OH is necessary for the addition of the next nucleotide. 02/24/2025 13  DEOXY VERSUS DIDEOXY 02/24/2025 MALLIGE COLLEGE OF PHARMACY 14 02/24/2025 MALLIGE COLLEGE OF PHARMACY 15  PRINCIPLE The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains. These chains terminating at specific nucleotide positions. Separated by gel electrophoresis Read DNA sequence 02/24/2025 MALLIGE COLLEGE OF PHARMACY 16  REQIREMENTS DNA sequencing is performed in four separate tubes, each containing i. Single stranded DNA to be sequenced ii. DNA polymerase iii. Primers iv. The four dNTPs (dATP, dCTP, dTTP and dGTP) v. Small amount of one of the four ddNTPs (ddATP or ddCTP or ddTTP or ddGTP) Either the primers or the dNTPs are radiolabeled with 32P 17 PROCEDURE DNA strand to be analyzed is used as a template and DNA polymerase is used, in a PCR reaction, to generate complimentary strands using primers. 4 different PCR 4 mixtures are prepared, each containing a certain percentage of ddNTP analogs to one of the four nucleotides Synthesis of the new DNA strand continues until one of these analogs is incorporated, Each PCR reaction will end up containing a mixture of different lengths of DNA strands, all ending with the ddNTP. Electrophoresis is used to separate the strands of the 18 4 reactions, and determine the sequence of the Sequencing of DNA by the Sanger method Sample Output 1 lane  COMPARISON Sanger Method Maxam Gilbert Method Enzymatic Chemical Requires DNA Requires DNA synthesis Termination of chain Breaks DNA at different elongation nucleotides Automation Automation is not available Single-stranded Double-stranded or DNA. single- stranded DNA 02/24/2025 MALLIGE COLLEGE OF PHARMACY 22  Applications of DNA Sequencing Forensics – Identify individuals – Determine the paternity of a child – Identifies endangered and protected species Medicine – Detect genes that are hereditary or cause diseases Agriculture – Map the genome of microorganisms 02/24/2025 23  THANK YOU 02/24/2025 MALLIGE COLLEGE OF PHARMACY 24