핵산 염기서열 분석(DNA SEQUENCING)

Slides:

Advertisements

Similar presentations

PCR-technique Applications by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project.

Advertisements

Sequencing Using DNA dependant DNA polymerase Initiation & elongation commences Denature & anneal labelled primer * * dCTP dGTP dATP dTTPdCTP dATP dTTPdATP.

DNA Sequencing.

DNA sequencing Matt Hudson. DNA Sequencing Dideoxy sequencing was developed by Fred Sanger at Cambridge in the 1970s. Often called “Sanger sequencing”.

Chapter 19 (part 2) Nucleic Acids. DNA 1 o Structure - Linear array of nucleotides 2 o Structure – double helix 3 o Structure - Super-coiling, stem- loop.

Cycle Sequencing. Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis To characterize.

DNA Sequencing How do you do it?. DNA Sequencing DNA sequencing – used to determine the actual DNA sequence of an organism. Using a computer, one can.

Nucleotides and Nucleic Acids Part 2 (and a little bit from Chapter 9) Chapter 8.

DNA Sequencing.

Chapter 10 DNA Sequencing.

More Molecular Genetic Technologies (Chapters 8, 9, 10) 1.Polymerase Chain Reaction (PCR) 1.Standard PCR 2.Real-time quantitative PCR 2.DNA sequencing.

Sanger-Coulson Dideoxynucleotide Sequencing Kwamina Bentsi-Barnes Deisy Mendoza Jennifer Aoki Lecture 10/30/00 Best printed in color for clarity.

7.1 cont’d: Sanger Sequencing SBI4UP MRS. FRANKLIN.

DNA Sequencing. * Sequencing means finding the order of nucleotides on a piece of DNA. * Nucleotide order determines amino acid order, and by extension,

DNA Sequencing Chemical Method and Termination Method Shaila Ahmed 02/13/04 BICM

DNA Sequencing Today, laboratories routinely sequence the order of nucleotides in DNA. DNA sequencing is done to: Confirm the identity of genes isolated.

1.) DNA Extraction Follow Kit Grind sample Mix with solution and spin Bind, Wash, Elute.

Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA tells size tells which tissues or conditions.

6.3 Advanced Molecular Biological Techniques 1. Polymerase chain reaction (PCR) 2. Restriction fragment length polymorphism (RFLP) 3. DNA sequencing.

Omar Alomair Homod Alherbi Fisal Alkulify

DNA sequencing: Importance Basic blueprint for life; Aesthetics. Gene and protein. –Function –Structure –Evolution Genome-based diseases- “inborn errors.

Announcements Lab notebooks due Monday by 5 No Ch. 9 Part 2 homework

A Lot More Advanced Biotechnology Tools (Part 1) Sequencing.

1 Chapter 2: DNA replication and applications DNA replication in the cell Polymerase chain reaction (PCR) Sequence analysis of DNA.

More Molecular Genetic Technologies (Chapters 8, 9, 10)

Restriction Fragments and Mapping Restriction Fragment Analysis – System used to compare the genes and DNA sequences between individuals in a population.

CHAPTER 7 DNA SEQUENCING - INTRODUCTION - SANGER DIDEOXY METHOD - AUTOMATED SEQUENCING - NEXT GENERATION OF SEQUENCING METHODS MISS NUR SHALENA SOFIAN.

DNA Sequencing Scenario

Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition.

Biotechnology -- Chap. 16. The use of biological systems for the production of materials (most work is in the field of Genetic Engineering)

DNA Sequencing.

6.3 Advanced Molecular Biological Techniques 1. Polymerase chain reaction (PCR) 2. Restriction fragment length polymorphism (RFLP) 3. DNA sequencing.

Sequencing by the Sanger Dideoxynucleotide Chain Termination Method 1. Prepare replication template denature, add synthetic primer, promote annealing TAGGCGA.

Genomics Part 1. Human Genome Project  G oal is to identify the DNA sequence of every gene in humans Genome  all the DNA in one cell of an organism.

DNA Sequencing Mimi Chen & Joanne Kim

DNA Sequencing Mimi Chen & Joanne Kim

DNA Sequencing Sanger Di-deoxy method of Sequencing Manual versus Automatic Sequencing.

DNA Sequencing A population of DNA fragments is generated. –One end is common to all fragments (the 5’ end of the sequencing primer). –The other end terminates.

Higher Human Biology Unit 1 Human Cells KEY AREA 5: Human Genomics.

중합효소연쇄반응 (PCR) 핵산 염기서열 분석(DNA SEQUENCING)

CATEGORY: EXPERIMENTAL TECHNIQUES Polymerase Chain Reaction (PCR) Tarnjit Khera, University of Bristol, UK Background The polymerase chain reaction (PCR)

Topic Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert.

The stroke size should be 0.25

Introduction to Microbiology Lecture 4

DNA sequencing DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology.

DNA sequencing © 2016 Paul Billiet ODWS.

Restriction Fragments and Mapping

DNA Sequencing BCH 446.

DNA Sequencing Techniques

Di-deoxynucleotide Chain Termination

Genetic Research and Biotechnology

Sequencing Technologies

DNA Sequencing Chemical Method and Termination Method

DNA sequencing Direct determination of nucleotide sequence

The Human Genome Project

DNA Sequence Determination (Sanger)

Polymerase Chain Reaction (PCR)

Screening a Library for Clones Carrying a Gene of Interest

UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia

DNA Sequencing A population of DNA fragments is generated.

DNA Sequencing A population of DNA fragments is generated.

5. DNA Sequencing pp

Molecular Biology lecture -Putnoky

Sequencing DNA – the Sanger Method – the most-popular way (one of many methods) A primer binds to one of the DNA strands at a specific location (such as.

Polymerase Chain Reaction (PCR) & DNA SEQUENCING

Plant Biotechnology Lecture 2

DNA Sequencing.

SBI4U0 Biotechnology.

Introduction, Chemistry, Protocols & Troubleshooting

Polymerase Chain Reaction (PCR) & DNA SEQUENCING

Presentation transcript:

핵산 염기서열 분석(DNA SEQUENCING)

핵산의 염기서열 분석 (Nucleotide sequencing) RNA sequencing DNA sequencing methods 1977 Sanger & Coulson at MRC Centre, UK DNA sequencing with chain-terminating inhibitors (효소작용과 사슬 종결법) 1977 Gilbert & Maxam at Harvard Univ. DNA sequencing by chemical degradation (말단 표지와 화학적 분해법) Automatic sequencer Next-generation (Next-Gen) sequencing methods

Sanger method Chain termination method (사슬 종결법) Based on the selective incorporation of chain- terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

NTP, dNTP, ddNTP

DNA replication 3’-OH 말단에 다음 nucleotide의 5’-PO4가 phosphodiester 결합 dNTP (deoxyribonucleoside triphosphate) 3’-OH 있음 다음 nucleotide 결합 가능 복제 진행 ddNTP (dideoxyribonucleoside triphosphate) 3’-OH 없음 다음 nucleotide 결합 불가 복제 (사슬) 종결

Sanger method의 특징 DNA 복제 중 특정 위치에서 사슬 종결 부분적 종결 유도 dNTP와 ddNTP의 비율 조절 4 Rxs : ddATP, ddTTP, ddGTP, ddCTP 종결 위치에 따라 복제된 DNA 사슬의 길이가 다름 : 전기영동으로 분리 부분적 종결 유도 dNTP와 ddNTP의 비율 조절 ex) (dATP : ddATP) 조절 dTTP, dGTP, dCTP는 충분히

사슬 종결법에 필요한 것들 Target DNA ssDNA dsDNA : 가닥 분리(변성) 후 primer 부착 Primer Klenow fragment dNTP (dATP, dTTP, dGTP, dCTP) ddNTP (ddATP, ddTTP, ddGTP,ddCTP) labeled with fluorescent material (different colors)

Dideoxyribonucleotide Chain Termination 1. Denaturation of Target DNA : dsDNA 2. Primer annealing 3. Radio-labeling by DNA replication Klenow fragment 35S-labeled dCTP* 4. Dideoxyribonuclotide Chain Termination Divide into FOUR (ddATP, ddTTP, ddGTP, ddCTP) Chain elongation & Chain termination 5. Electrophoresis & Autoradiography Polyacrylamide gel electrophoresis (Capillary) X-ray film (Fluorescent, Laser) 6. Reading : from the short one

핵산 염기서열 분석의 이용 Molecular biology study genomes and the proteins they encode identify new genes, associations with diseases & phenotypes Medicine determine the risk of genetic diseases identify potential drug targets Forensics forensic identification, parental testing Evolutionary biology how different organisms are related how different organisms evolved Metagenomics identification of unculturable organisms important in ecology, epidemiology, microbiology, …

Automated DNA Sequencing Automated DNA Sequencer fluorescent labeling capillary electrophoresis general automation (computer, etc) used the Sanger sequencing method Human Genome Project (2001)

Next-Gen Sequencing methods Massively parallel signature sequencing (MPSS) Polony sequencing 454 pyrosequencing Illumina (Solexa) sequencing SOLiD sequencing Ion Torrent semiconductor sequencing DNA nanoball sequencing Heliscope single molecule sequencing Single molecule real time (SMRT) sequencing Nanopore DNA sequencing Tunneling currents DNA sequencing Sequencing by hybridization in vitro virus high-throughput sequencing …