DNA Technologies (Introduction) Cloning PCR Gel Electrophoresis DNA Sequencing DNA fingerprinting GMO : Genetically Modified Organism ……

Biotechnology Biotechnology Any technique applied to biological systems to manipulate processes DNA technologies isolate purify, analyze and manipulate DNA sequences DNA fingerprinting used in forensics Genetic engineering uses DNA technologies to alter genes for practical purposes

DNA Cloning Bacterial enzymes (restriction endonucleases) form the basis of DNA cloning Bacterial plasmids illustrate the use of restriction enzymes in cloning DNA libraries contain collections of cloned DNA fragments Polymerase chain reaction (PCR) amplifies DNA in vitro

Recombinant DNA DNA cloning provides many copies of a gene Used for research or manipulation Recombinant DNA contains DNA from multiple sources joined together Recombinant plasmids containing gene of interest can be cloned in E. coli

Cloning DNA Fragments

interest from cells and cut the DNA into fragments. Gene of interest Plasmid from bacterium Cell 1 Cut a circular bacterial plasmid to make it linear. 2 Isolate genomic DNA containing gene of interest from cells and cut the DNA into fragments. Figure 18.2 Overview of cloning DNA fragments in a bacterial plasmid. Insert the genomic DNA fragments into the plasmid to make recombinant DNA molecules. Recombinant DNA is DNA from two different sources joined together. Here, the recombinant DNA molecules are the recombinant plasmids. 3

Introduce recombinant molecules into bacterial cells; each bacterium Inserted genomic DNA fragment Recombinant DNA molecules Introduce recombinant molecules into bacterial cells; each bacterium receives a different plasmid. As the bacteria grow and divide, the recombinant plasmids replicate, thereby amplifying the piece of DNA inserted into the plasmid. 4 Bacterium Bacterial chromosome Progeny bacteria 대부분의 경우는 원하는 gene이 없음. Identify the bacterium containing the plasmid with the gene of interest inserted into it. Grow that bacterium in culture to produce large amounts of the plasmid for experiments with the gene of interest. 5

Restriction Enzyme EcoRI Restriction site for EcoRI DNA 1 EcoRI restriction enzyme cleaves sugar–phosphate backbones at arrows. Sticky end Sticky end DNA fragments with the same sticky ends can pair. Shown here is a DNA fragment inserting between two other DNA fragments, as happens when inserting a DNA fragment into a bacterial plasmid. 2 Another DNA fragment produced by EcoRI digestion 제한효소와 ligase를 이용하여 recombination 완성  어느정도 control 되고 있음. 자를 때와 붙일 때 palindrom sequence가 유리... Nick in sugar–phosphate backbone Nicks in sugar– phosphate backbones are sealed by DNA ligase. 3 Recombinant DNA molecule

Endonucleases (Restriction Enzymes) Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites Restriction fragments result Sticky ends have unpaired bases at cuts which will hydrogen bond Ligase stitches together paired sticky ends

Plasmid Cloning Gene of interest Restriction site lacZ+ gene Cell ampR vector DNA fragments with sticky ends Cut plasmid cloning vectors with a restriction enzyme to produce sticky ends Stick end : 점착성 말단

Plasmid Cloning No transfection No recombination Inserted DNA fragments with gene of interest Inserted DNA fragment without gene of interest Resealed plasmid cloning vector with no inserted DNA fragment Recombinant plasmids Nonrecombinant plasmid 빨간색 No transfection No recombination

Untransformed bacterium containing ampicillin. Plasmid Cloning Bacteria not transformed with a plasmid Bacteria transformed with plasmids Selection: Transformed bacteria grow on medium containing ampicillin because of ampR gene on plasmid. Untransformed bacterium cannot grow on medium containing ampicillin. Screening: Blue colony contains bacteria with a non-recombinant plasmid; that is, the lacZ+ gene is intact. Plate containing ampicillin and X-gal 그래도 확실하지 않다…  추가적인 실험이 필요함. White colony contains bacteria with a recombinant plasmid; that is, the vector with an inserted DNA fragment. Once the white colony with the gene of interest is identified, it can be grown in culture to produce large quantities of the plasmid. lacZ(가수분해) X-gal(white) Blue

Plasmid Cloning Vectors Engineered to contain gene of interest and sorting genes Sorting genes identify E. coli with cloned plasmid  E. coli with appropriate plasmid are 1. ampicillin resistant (transformation check by survival) 2. and blue-white screened on X-gal (recombination check by color change)  then, DNA hybridization (probe) finds the gene of interest

DNA Hybridization Uses nucleic acid probe to identify gene of interest in set of clones Probe has tag (표지) for detection Identified colony produces large quantities of cloned gene Tag: radioactive or fluroscent

DNA Hybridization

Single strand, 흡착 Bacterial Culture medium containing ampicillin colony Filter paper Replica of bacterial colonies Filter paper Single strand, 흡착

Labeled probe (single stranded) Plasmid DNA (single stranded) Bag Labeled single-stranded DNA probe for the gene of interest Bag Filter Hybridization has occurred between the labeled probe and the plasmids released from the bacteria in this colony. The hybridization is detected in subsequent steps. DNA probe:

Corresponds to Developed one colony on photographic master plate film Figure 18.5: Research Method. DNA Hybridization to Identify a DNA Sequence of Interest Original master plate

Polymerase Chain Reaction Polymerase chain reaction (PCR) Produces many sequence copies without host cloning Amplifies known DNA sequences for analysis Only copies sequence of interest Primers bracket sequence Agarose gel electrophoresis Separates fragments by size and charge Gel molecular sieve

Polymerase Chain Reaction Cycle 1 Cycle 2 Cycle 3 2 molecules produced Produces 4 molecules Produces 8 molecules Target sequence Template DNA primers DNA containing target sequence to be amplified DNA primer New DNA These 2 molecules match target DNA sequence DNA primer New DNA Target sequence Target sequence Template In solution, target sequence(2), DNA primer(2), free nucleotides, Taq DNA – polymerase

Agarose Gel Electrophoresis 항상

DNA Sequencing Used for small DNA sequences to genomes Dideoxy (Sanger) method of sequencing Dideoxyribonucleotides have –H bound to 3’ C instead of –OH DNA polymerases place dideoxyribonucleotides in DNA, stops replication Polyacrylamide gel separates strands varying by one nucleotide

Dideoxy (Sanger) Method

Deoxyribonucleo-tide precursors Dideoxyribonucleotide (dd) precursors DNA strand to be sequenced DNA polymerase DNA primer dATP ddATP Deoxyribonucleo-tide precursors Dideoxyribonucleotide (dd) precursors (fluorescently labeled) Figure 18.18: Research Method. Dideoxy (Sanger) Method for Sequencing DNA

DNA to be sequenced Insertion of dideoxyribonucleotide stops synthesis Primer bound to template New DNA synthesis in 5' 3' direction An electrophoretic gel Dye-labeled fragments of DNA migrating through the gel Detector registers fluorescence from DNA as laser beam hits them Laser Laser beam pass through gel Visualization of the DNA sequence Sequence obtained from experiment

Whole-Genome Shotgun Sequencing Genomic DNA DNA fragments Genomic DNA fragment and so on Plasmid cloning vector DNA sequence of genomic DNA fragment (actual sequence is several hundred base pairs) Assembled sequence