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Biotechnologies DNA Technologies Overview Natalia Volodina, Ph.D. CHAPTER 9 DNA Technologies Key topics: – DNA cloning techniques – DNA analysis methods – DNA microarray technology – Expression of recombinant proteins Recombinant DNA • Artificially created DNA that combines sequences that do not occur together in the nature • Basis of much of the modern molecular biology – Molecular cloning of genes – Over-expression of proteins – Transgenic food, animals … DNA Cloning • Organism cloning: – Creation of identical copies of an organism • DNA cloning: – Creation of identical copies of a piece of DNA (gene) – isolate a specific gene from the source organism and amplify it in the target organism • Basic steps: – – – – – Cut the source DNA at the boundaries of the gene Select a suitable carrier DNA (vector) Insert the gene into the vector Insert the recombinant vector into host cell Let the host produce multiple copies of recombinant DNA DNA Cloning: General Scheme Restriction Endonucleases • Cleave DNA phosphodiester bonds at specific sequences • Common in bacteria – Eliminates infectious viral DNA • Some make staggered cuts – Sticky ends • Some make straight cuts – Blunt ends • Large number are known: – Commercially available – Well documented: REBASE – http://rebase.neb.com/rebase/rebase.html Cloning Vectors • Plasmids – Circular DNA molecules that are separate from the bacterial genomic DNA – Can replicate autonomously – Carry antibiotic resistance genes – Allows cloning of DNA up to 15,000 bp • Bacteriophage l – Virus that infects bacteria – Efficient delivery of DNA – Allows cloning of DNA up to 23,000 bp DNA Ligase • Enzyme that covalently joins two DNA fragments – Normally function in DNA repair – Human DNA ligase uses ATP – Bacterial DNA ligase uses NAD Antibiotic Selection • Antibiotics, such as penicillin and ampicillin, kill bacteria • Plasmids can carry genes that give host bacterium a resistance against antibiotics • Allows growth (selection) of bacteria that have taken up the plasmid OH O O N S H O N H H N H Identification of Empty Plasmids Colony Lifts • Bacterial colonies can grow on agar plates • Each colony is made of descendants of a single bacterium—cells within a colony are clones • Colonies may differ from each other • Colonies can be lifted from the agar plate to the nitrocellulose paper • Lifted cells can be broken up by alkali treatment Detection of Specific DNA Sequences • Upon cell lysis, DNA (and cellular proteins) stick to the paper • Specific probes can be used to detect the presence of specific macromolecules • To detect specific DNA, a radiolabeled DNA probe, complementary to the target sequence, is used. • How would you detect specific proteins? Polymerase Chain Reaction: Steps • Allows to amplify DNA in the test tube – Can amplify regions of interests (genes) within a linear DNA – Can amplify complete circular plasmids • Mix together – Target DNA – Primers (oligos complementary to target) – Nucleotides: dATP, dCTP, dGTP, dTTP – Thermostable DNA polymerase • Place the mixture into thermocycler – Melt DNA at about 95 ºC – Cool separated strands to about 50-60 ºC – Primers anneal to the target – Polymerase extends primers in 5’3’ direction – After a round of elongation is done, repeat steps Outline of PCR Site-Directed Mutagenesis • Understanding the function of proteins often requires that a specific amino acid residue be mutated • To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene • Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA Separation of DNA by Electrophoresis • Negatively charged DNA migrates to the anode in the presence of an electric field • Agarose gel hinders the mobility of DNA molecules • Mobility depends on the size and the shape – Small molecules faster – Compact molecules faster • Practical use is – DNA analysis – DNA purification – DNA-protein interaction studies DNA Sequence Analysis • Sequencing – Chain termination method (Sanger sequencing) – Sequencing by synthesis (Pyrosequencing) • Single Nucleotide Polymorphism – Molecular beacons Genomic Sequencing DNA Fingerprinting • Each individual has a unique DNA sequence • Differences cause variations in the length of fragments that form when sample is treated with restriction enzymes – RFLP stands for restriction fragment length polymorphism • Fragments can be separated by size using agarose gel electophoresis • Separated DNA fragments can be blotted on the membrane (Southern blot) • Few of the blotted DNA fragments are visualized by hybridizing with a labeled nucleic acid probe • Allows matching “suspect” samples to known individuals The Southern Blot Analysis of RFLP Expression of Cloned Genes • We want to study the protein product of the gene • Special plasmids, called expression vectors, contain sequences that allow transcription of the inserted gene • Expression vectors differ from cloning vectors by having – Promoter sequences – Operator sequences – Code for ribosome binding site – Transcription termination sequences Purification of Recombinant Genes • Purification of natural proteins is difficult • Recombinant proteins can be tagged for purification • The tag binds to the affinity resin and thus captures the fusion protein – GST – His-Tag Eukaryotic Gene Expression in Bacteria • An eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium • Eukaryotic genes have – Exons: coding regions – Introns: noncoding regions • Introns in eukaryouric gene pose problems • Bacteria cannot splice introns out • mRNA is intron-free genetic material Construction of cDNA • mRNA can be extracted from eukaryotic cells • All mRNA molecules have poly-A tail – helps in purification of mRNA – serves as an universal template • DNA strand can be synthesized using mRNA as a template • This is catalyzed by the reverse transcriptase • The end result is a hybrid where the DNA strand is complementary to the mRNA • The hybrid can be converted to duplex DNA, known as cDNA DNA Microarrays: Applications DNA Microarrays allow simultaneous screening of many thousands of genes: high-throughput screening • genome wide genotyping – Which genes are present in this individual? • tissue-specific gene expression – Which genes are used to make proteins? • mutational analysis – Which genes have been mutated? DNA Microarrays: Design Two fundamental approaches • One-color array – – – – – • Patented and commerialized by Affymetrix Photolitographic synthesis of probe DNA on the chip Targets are biotin labeled Bound targets detected using streptavidin-fluorofore complex Widely used in industry Two-color array – – – – Developed by Stanford University, 1996 Probes sometimes pipetted on the chip Targets linked to either green or red fluorescent labels Used often in academia One-color Quantitative Microarray Technology • General Process – – – – Target is biotin-labeled cRNA Probe is single-stranded DNA oligo attached to the wafer Complementary target and probe hybridize Duplex stained with the streptavidin-bound fluorescent marker • In situ oligonucleotide synthesis (Affymetrix U.S. patent 5,861,242 ) – 5-inch square quartz wafer with covalently bound layer of silane – Parallel synthesis with photolithographic masks – Makes oligos 20-25 nucleotides long; 400,000 per chip Photolitographic Synthesis of DNA GeneChip®: Detection • Bound nucleic acid duplexes have a covalently attached biotin • Biotin binds a streptavidin-phycoerythrin complex • Phycoerythrin is a dye from red algae – Absorbs blue light at 488 nm – Gives off fluorescent light at about 600 nm • Fluorescence intensity is proportional to concentration of bound nucleic acid duplexes O N H HOOC COOH N H N N H Phycoerythrin (pigment in red alga) O Two-Color DNA Microarray “Genome Chips” Fluorescent Spots in Two-color DNA Microarray Chapter Summary In this chapter, we learned how: • • • • • • to make recombinant DNA to use bacteria for DNA cloning to analyze DNA by size and sequence to mutate and amplify DNA in test-tube to express and purify eukaryotic genes to determine expression levels of genes