10. Monoclonal antibody technology. 8. DNA microarray technology 9. Human Genome Project. 12. RNA interference (RNAi) 11. Antisense technology

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1 Topics 1. Recombinant DNA technology 2. DNA cloning 3. DNA library 4. Southern blotting 5. RFLP 6. DNA amplification: PCR 7. DNA sequencing 8. DNA microarray technology 9. Human Genome Project 10. Monoclonal antibody technology 11. Antisense technology 12. RNA interference (RNAi) 13. Epigenetics 14. Other topics Basic Principles of Recombinant DNA Technology Cutting and Joining DNA Restriction endonucleases act as scissors at specific sites by breaking covalent bonds. Restriction fragments EcoRI: G-AATTC, sticky ends HindIII: A-AGCTT, sticky ends BamHI: G-GATCC, sticky ends AluI: AG-CT, blunt ends SmaI: CCC-GGG, blunt ends EcoRI restriction enzyme cleavage produces "sticky" ends. SmaI restriction enzyme cleavage produces blunt" ends. Restriction Enzymes Enzyme Source EcoRI Escherichia coli Recognition Sequence 5'GAATTC 3'CTTAAG Cut 5'---G AATTC---3' 3'---CTTAA G---5' BamHI Bacillus amyloliquefaciens 5'GGATCC 3'CCTAGG 5'---G GATCC---3' 3'---CCTAG G---5' HindIII Haemophilus influenzae 5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3'---TTCGA A---5' TaqI Thermus aquaticus 5'TCGA 3'AGCT 5'---T CGA---3' 3'---AGC T---5' NotI Nocardia otitidis 5'GCGGCCGC 3'CGCCGGCG 5'---GC GGCCGC---3' 3'---CGCCGG CG---5' HinfI Haemophilus influenzae 5'GANTC 3'CTNAG 5'---G ANTC---3' 3'---CTNA G---5' Sau3A Staphylococcus aureus 5'GATC 3'CTAG 5'--- GATC---3' 3'---CTAG ---3' PovII* Proteus vulgaris 5'CAGCTG 3'GTCGAC 5'---CAG CTG---3' 3'---GTC GAC---5' SmaI* Serrana mauceceus 5'CCCGGG 3'GGGCCC 5'---CCC GGG---3' 3'---GGG CCC---5' HaeIII* Haemophilus egytius 5'GGCC 3'CCGG 5'---GG CC---3' 3'---CC GG---5' AluI* Arthrobacter luteus 5'AGCT 3'TCGA 5'---AG CT---3' 3'---TC GA---5' EcoRV* Escherichia coli 5'GATATC 3'CTATAG 5'---GAT ATC---3' 3'---CTA TAG---5' * = blunt ends

2 Palindrome, Restriction Enzyme, Sticky Ends Arber, Nathans, Smith (1978) CIVIC, Madam Sticky Ends (Cohesive Ends) GAATTC G AATTC G AATTC GAATTC G AATTC G AATTC Get An Apple To The Class Juang RH (2004) BCbasics Basic Principles of Recombinant DNA Technology Cutting and Joining DNA DNA ligases glue DNA fragments. Sticky ends/blunt ends No discrimination between DNAs with different origins DNA cloning: cut, paste, and transfer to a host cell (vector) for amplification Separating Restriction Fragments and Visualizing DNA Gel electrophoresis Sugar-phosphate backbone is negatively charged. Larger molecules move more slowly than smaller molecules. Resolution is dependent upon the percentage amount of agarose. The rate at which these linear fragments of DNA migrate is inversely proportional to the log10 of the molecular weight of the fragment.

3 Restriction Mapping of DNA Restriction enzymes CK A B A+B M A B 10 kb A 8 kb 2 kb B 7 kb 3 kb A +B 5 kb 3 kb 2 kb Juang RH (2004) BCbasics DNA electrophoresis apparatus. An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates towards the camera.

4 Restriction digest of plasmid DNA from Escherichia coli, run on a 1% agarose gel and stained with ethidium bromide. A Kb DNA ladder Uncut plasmid DNA A single digest ion of the plas mid with EcoRI A single diges tion with XhoI A double diges tion - both Eco RI and XhoI The Specific Cutting and Ligation of DNA GAATTC GAATTC CTTAAG CTTAAG G AATTC G AATTC CTTAA G CTTAA G G G AATTC AATTC CTTAA CTTAA G G G AATTC CTTAA G Juang RH (2004) BCbasics DNA Cloning 1. Isolation of DNA 2. Ligation of the DNA into a vector 3. Transformation of a host cell with the recombinant DNA 4. Selection of host cells harboring the recombinant DNA 5. Screening of cells for those harboring the recombinant DNA or producing the appropriate protein product

5 The plasmid pbr322 is one of the most commonly used E.coli cloning vectors. leicacids/mappbr322.htm

6 Cloning Vectors Bacterial Vectors Plasmids pbr The molecule is small so that the vector can accommodate DNA of up to 5 to 10 kb. 2. Several unique restriction sites for inserting a DNA fragment 3. Genes encoding resistance to ampcillin and tetracycline for insertional inactivation of a selectable marker puc18/puc19 The uninterrupted lacz gene produces an enzymatically active β-galactosidase (blue). Figure 3.9 puc18.txt Cloning Vectors Vector Insert size Plasmids 10 kb Bacteriophage Bacteriophage 5-20 kb Cosmids Cosmids kb Yeast Artificial Chromosomes (YAC) Bacterial Artificial Chromosomes (BAC) YAC kb Plant Cloning Vectors BAC kb Mammalian Cell Vectors - Retroviruses: single-strand RNA virus (reverse transcriptase) - Adenoviruses: double-strand DNA virus Cell transformation Transformation: transferring DNA into cells Bacteria: heat shock in the presence of DNA Eukaryotes: - Plants, fungi, algae degradation of cell walls to produce protoplasts followed by electroporation biolistics: gene/dna gun, W/Au/Ag particles viruses - Animal cells electroporation viruses microinjection

7 Constructing and Screening a DNA Library DNA libraries: a collection of DNA fragments produced by restriction enzymes and inserted into plasmids or other cloning vectors such as viruses Genomic library: DNA fragments of the entire genome of an organism (Figure 3.15) Complementary DNA (cdna) library: derived from mrna of a tissue, includes only sequences expressed at a given time in a specific tissue, i.e. only expressed genes from a certain type of cell. (Figure 3.16) The expression of cdnas in recombinant cells can be used to produce large quantities of proteins in vitro. - no non-coding introns - no genome that does not codes for RNA (non-coding DNA) - no regulatory elements associated with genes (eg. promoters, enhancers)

8 Southern Blot Hybridization: Gel Transfer Detection of specific DNA fragments by gel-transfer hybridization (Southern blotting). (A) The mixture of double-stranded DNA fragments generated by restriction nuclease treatment of DNA is separated according to length by electrophoresis. (B) A sheet of either nitrocellulose paper or nylon paper is laid over the gel, and the separated DNA fragments are transferred to the sheet by blotting. The gel is supported on a layer of sponge in a bath of alkali solution, and the buffer is sucked through the gel and the nitrocellulose paper by paper towels stacked on top of the nitrocellulose. As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet, where they adhere firmly. This transfer is necessary to keep the DNA firmly in place while the hybridization procedure (D) is carrried out. (C) The nitrocellulose sheet is carefully peeled off the gel. (D) The sheet containing the bound single-stranded DNA fragments is placed in a sealed plastic bag together with buffer containing a radioactively labeled DNA probe specific for the required DNA sequence. The sheet is exposed for a prolonged period to the probe under conditions favoring hybridization. (E) The sheet is removed from the bag and washed thoroughly, so that only probe molecules that have hybridized to the DNA on the paper remain attached. After autoradiography, the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph. An adaptation of this technique to detect specific sequences in RNA is called Northern blotting. In this case mrna molecules are electrophoresed through the gel and the probe is usually a single-stranded DNA molecule.

9 RFLP DNA from different individuals varies in the number and locations of sites that react with different restriction enzymes and with different probes. One individual can have restriction sites that do not appear in another individual. When DNA from different individuals is subjected to restriction enzymes, gel electrophoresis, and probes, different patterns emerge. This method can be used to establish the identity of individuals who have left samples of their DNA at a crime scene, to establish paternity, or to exonerate people who have been accused of a crime.

10 DNA_DetectivePC.exe

11 DNA Amplification PCR.exe

12 Taq polymerase Originally isolated by Thomas D. Brock in 1965 from the thermophilic bacterium Thermus aquaticus Taq's temperature optimum for activity is 75-80C, with a halflife of 9 minutes at 97.5C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72C. One of Taq's drawbacks is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity[4], and has an error rate measured at about 1 in 9,000 nucleotides. Pfu DNA polymerase: possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification Protein model for Taq polymerase DNA Sequencing cycseqpc.exe

13 How DNA Sequence Is Determined? Polyacrylamide Gel Electrophoresis DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis 32 P 32 P 32 P 32 P 32 P 32 P 32 P 32 P 32 P 32 P If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence But these bands can t tell us the identity of the terminal nucleotides Juang RH (2004) BCbasics 1 5 P R P R P R P R P R P R 5 A How to Obtain DNA Fragments 1 Maxam-Gilbert's Method: 32 P 32 P 32 P Terminated Specific Reaction to G Sanger's Method: 32 P Biosynthetic method 32 P STOP 2 Chemical method Non-radioactive (invisible) Terminated Keep on going ddntp Destroy Cleavage Destroy Cleavage 3 Analogue Template Producing various fragments Juang RH (2004) BCbasics 5 A Normal Linking 2 Phosphodiester bond 3 3 Sanger s Method: How Terminated Can not react dideoxynuceotide Juang RH (2004) BCbasics

14 Sanger-Coulson Sequencing Method : chain termination method using single-stranded (ss) DNA Maxam-Gilbert s DNA Sequencing Method : chemical cleavage method using double-stranded (ds) DNA : purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA 1. Radioactive labelling at the 5 end of a ds DNA to be sequenced 2. Separation and purification of the two strands of the DNA (one of the strands heavier than the other since it contains more purine nucleotides (A and G) than lighter pyrimidines (C and T)) 3. Each strand is treated chemically with cleavage reagents. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. 4. Fragments size-separated by gel electrophoresis, with the four reactions arranged side by side. 5. For visualization, the gel is exposed to X-ray film for autoradiography, yielding an image of a series of dark 'bands' corresponding to the radiolabelled DNA fragments. Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity, extensive use of hazardous chemicals, and difficulties with scale-up. In addition, unlike the chain-termination method, chemicals used in the Maxam-Gilbert method cannot easily be customized for use in a standard molecular biology kit. Example of a Sanger sequencing read The four bases are detected using different fluorescent labels. These are detected and represented as 'peaks' of different colours, which can then be interpreted to determine the base sequence. This read is from the Rms149 plasmid sequencing project, and used dye terminater chemistry. The re gion on the left with a cream background has been identified as vector sequence. Sequence ladder by radioactive sequencing compared to fluorescent peaks

15 DNA Sequencer Applied Biosystems Subsidiary of Applera National Institutes of Health Animation ( Timeline Genes, Variation and Human History How to Sequence a Human Genome Ethical, Legal and Social Implications (ELSI) Bioinformatics Exploring our Molecular Selves (3-D Animation) Different genome sizes Organism Genome size (base pairs) Virus, Bacteriophage MS First sequenced RNA-genome Virus, SV Virus, Phage Φ-X174; First sequenced DNA-genome Virus, Phage λ Bacterium, Carsonella ruddii Smallest non-viral genome, Feb 2007 Bacterium, Buchnera aphidicola Bacterium, Wigglesworthia glossinidia Bacterium, Escherichia coli Amoeba, Amoeba dubia Largest known genome, Dec 2005 Plant, Arabidopsis thaliana First plant genome sequenced, Dec 2000 Plant, Fritillaria assyrica Plant, Populus trichocarpa First tree genome, Sept 2006 Fungus,Saccharomyces cerevisiae Nematode, Caenorhabditis elegans First multicellular animal genome, Dec Insect, Drosophila melanogaster aka Fruit Fly Insect, Bombyx mori aka Silk Moth Insect, Apis mellifera aka Honey Bee Mammal, Homo sapiens Note: The DNA from a single human cell has a length of ~1.8 m, width of ~ 2.4 nm.

16 Genes and human genome A gene is a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions. The estimated number of genes in the human genome 20,488, with perhaps 100 more yet to be discovered Pennisi, 2007, "Working the (Gene Count) Numbers_ Finally, a Firm Answer". Science 316, p.1113 Gene density of a genome: a measure of the number of genes per million base pairs (Mb) Prokaryotic genomes have much higher gene densities than eukaryotes. The gene density of the human genome ~ genes/mb The number of human genes << that of many much simpler organisms. However, human cells make extensive use of alternative splicing to produce several different proteins from a single gene, and the human proteome is thought to be much larger than those of the aforementioned organisms. Most human genes have multiple exons. Human introns are frequently much longer than exons. In addition to protein coding genes, the human genome contains thousands of RNA genes, including trna, ribosomal RNA, microrna, and other non-coding RNA genes. Human genome overview: X chromosome s.cgi?taxid=9606&build=previous&chr=x

17 Bioinformatics: creation and advancement of algorithms, computational and statistical techniques, and theory to solve formal and practical problems arising from the management and analysis of biological data Computational biology: hypothesis-driven investigation of a specific biological problem using computers, carried out with experimental or simulated data, withthe primary goal of discovery and the advancement of biological knowledge Systems biology: systematic study of complex interactions in biological systems. - Study of the interactions between the components of biological systems, and how these interactions give rise to the function and behavior of that system (for example, the enzymes and metabolites in a metabolic pathway) Research areas of bioinformatics Analysis of gene expression: Such studies are often used to determine the genes implicated in a disorder: one might compare microarray data from cancerous epithelial cells to data from non-cancerous cells to determine the transcripts that are up-regulated and down-regulated in a particular population of cancer cells. Analysis of regulation: For example, promoter analysis involves the identification and study of sequence motifs in the DNA surrounding the coding region of a gene. These motifs influence the extent to which that region is transcribed into mrna. Analysis of protein expression: Analysis of mutations in cancer: Massive sequencing efforts are used to identify previously unknown point mutations in a variety of genes in cancer. New physical detection technology are employed, such as oligonucleotide microarrays to identify chromosomal gains and losses (called comparative genomic hybridization), and single nucleotide polymorphism arrays to detect known point mutations. These detection methods simultaneously measure several hundred thousand sites throughout the genome, and when used in high-throughput to measure thousands of samples, generate terabytes of data per experiment. Prediction of protein structure: homology is used to predict the function of a gene: if the sequence of gene A, whose function is known, is homologous to the sequence of gene B, whose function is unknown, one could infer that B may share A's function. In the structural branch of bioinformatics, homology is used to determine which parts of a protein are important in structure formation and interaction with other proteins. Research areas of bioinformatics Sequence analysis: DNA sequences of hundreds of organisms have been decoded and stored in databases. The information is analyzed to determine genes that encode polypeptides, as well as regulatory sequences. A comparison of genes within a species or between different species can show similarities between protein functions, or relations between species. Another aspect of bioinformatics in sequence analysis is the automatic search for genes and regulatory sequences within a genome. Not all of the nucleotides within a genome are genes. Within the genome of higher organisms, large parts of the DNA do not serve any obvious purpose. This so-called junk DNA may, however, contain unrecognized functional elements. Bioinformatics helps to bridge the gap between genome and proteome projects--for example, in the use of DNA sequences for protein identification. Genome annotation: annotation is the process of marking the genes and other biological features in a DNA sequence. Computational evolutionary biology: study of the origin and descent of species, as well as their change over time. Informatics has assisted evolutionary biologists in several key ways; trace the evolution of a large number of organisms by measuring changes in their DNA, rather than through physical taxonomy or physiological observations alone, more recently, compare entire genomes, which permits the study of more complex evolutionary events, such as gene duplication, lateral gene transfer, and the prediction of factors important in bacterial speciation, build complex computational models of populations to predict the outcome of the system over time track and share information on an increasingly large number of species and organisms Measuring biodiversity: Databases are used to collect the species names, descriptions, distributions, genetic information, status and size of populations, habitat needs, and how each organism interacts with other species. One very exciting potential of this field is that entire DNA sequences, or genomes of endangered species can be preserved, allowing the results of Nature's genetic experiment to be remembered in silico, and possibly reused in the future, even if that species is eventually lost. Research areas of bioinformatics Comparative genomics: establishment of the correspondence between genes (orthology analysis) or other genomic features in different organisms Modeling biological systems: both analyze and visualize the complex connections of these cellular processes. Artificial life or virtual evolution attempts to understand evolutionary processes via the computer simulation of simple (artificial) life forms. High-throughput image analysis: high-throughput and high-fidelity quantification and sub-cellular localization (high-content screening) morphometrics clinical image analysis and visualization determining the real-time air-flow patterns in breathing lungs of living animals quantifying occlusion size in real-time imagery from the development of and recovery during arterial injury making behavioral observations from extended video recordings of laboratory animals infrared measurements for metabolic activity determination Protein-protein docking: to address the question whether it is practical to predict possible proteinprotein interactions only based on these 3D shapes, without doing protein-protein interaction experiments

18 Human genome overview: X chromosome s.cgi?taxid=9606&build=previous&chr=x The Neandertal Genome A Draft Sequence of the Neandertal Genome, Green et al., Science 7 May 2010: Vol no. 5979, pp Targeted Investigation of the Neandertal Genome by Array-Based Sequence Capture, Burbano et al., Science 7 May 2010: Vol no. 5979, pp About Neandertals Neandertals (Homo neanderthalensis) are currently believed to be our closest evolutionary relatives. Although some researchers once thought they were our immediate ancestors in Europe, most now agree that Neandertals and modern humans most likely shared a common ancestor within the last 500,000 years, possibly in Africa. The morphological features typical of Neandertals first appear in the European fossil record about 400,000 years ago, with bones of full-fledged Neandertals showing up at least 130,000 years ago. They lived in Europe and western Asia, as far east as southern Siberia and as far south as the Middle East (see map), before disappearing from the fossil record about 30,000 years ago. Samples and sites from which DNA was retrieved Segments of Neandertal ancestry in the human reference genome. We examined 2825 segments in the human reference genome that are of African ancestry and 2797 that are of European ancestry. (A) European segments, with few differences from the Neandertals, tend to have many differences from other present-day humans, whereas African segments do not, as expected if the former are derived from Neandertals. (B) Scatter plot of the segments in (A) with respect to their divergence to the Neandertals and to Venter. In the top left quandrant, 94% of segments are of European ancestry, suggesting that many of them are due to gene flow from Neandertals.

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20 Antigen Immunization Adapted from Milstein (1980) Scientific American, Oct. p m m m m Spleen cells BALB/c B cell Myeloma Cell fusion x Antiseum A mixture of all Ab Monoclonal antibodies Pure single Ab Each If a single B cell B produces cell was picked only one up kind and cultured, of antibody, then which it will binds produce to its only specific one kind antigen. of antibody. After Myeloma Each If B cell immunization, hybridoma is cell fused can with line be the can myeloma, cultured mouse produce in spleen the pure fused test contains single tube, cell might antibody, but B cells can be not producing cultured called produce monoclonal and specific useful produce antibodies. antibody. Conventional But B cell can antiserum not survive is well the mixture in the culture. of all antibodies produced by B cells from spleen.

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22 13.4 Medical Applications Production of Therapeutic Proteins Insulin, human growth hormone, Commercial synthesis of proteins: 1. Find an organism that is capable of producing the proteins. - purification, safety, contamination 2. An efficient production organism be genetically engineered to produce the desired protein. - bacteria (E. coli and Bacillus subtilis) - yeast fungi (S. cerevisiae, Neuspora crassa, and Aspergillus nidulans) : fast growth rates, simple and well-defined nutrient requirements : DNA transfer transformation and electroporation : bacteria no introns (mrna cdna), different codon usage, modifications of the protein structure can occur during posttranslational processing. - animal cell cultures : very expensive : DNA transfer microinjection, viral infection, and bombardment of cells with DNA-coated particles Various modes of alternative splicing Alternative splicing Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript, the pre-mrna, are separated and reconnected so as to produce alternative ribonucleotide arrangements. These linear combinations then undergo the process of translation where specific and unique sequences of amino acids are specified, resulting in isoform proteins. In this way, alternative splicing uses genetic expression to facilitate the synthesis of a greater variety of proteins. Known modes of alternative splicing: Alternative selection of promoters: Alternative selection of cleavage/polyadenylation sites: Intron retaining mode: Exoncassettemode It invalidates the old theory of one DNA sequence coding for one polypeptide (the "one-gene-oneprotein" hypothesis). t has been proposed that for eukaryotes it was a very important step towards higher efficiency, becauseinformationcanbestoredmuchmoreeconomically.severalproteinscanbeencodedina DNA sequence whose length would only be enough for two proteins in the prokaryote way of coding. Others have noted that it is unnecessary to change the DNA of a gene for the evolution of a new protein. Instead, a new way of regulation could lead to the same effect, but leaving the code for the established proteins unharmed.