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Exploring Genes and Genomes

Basic Tools of Gene Exploration. Restriction EndonucleasesDNA manipulationBlotting TechniquesSeparate and characterize DNADNA SequencingGene architecture; gene expression, protein structureSolid-Phase Synthesis of Nucleic AcidsIdentification and amplification of other nucleic acidsPolymerase

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Exploring Genes and Genomes

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    1. Chapter 5 Exploring Genes and Genomes

    2. Basic Tools of Gene Exploration Restriction Endonucleases DNA manipulation Blotting Techniques Separate and characterize DNA DNA Sequencing Gene architecture; gene expression, protein structure Solid-Phase Synthesis of Nucleic Acids Identification and amplification of other nucleic acids Polymerase Chain Reaction Amplification of nucleic acid

    3. Restriction Endonucleases Recognize specific base sequences in duplex DNA Palindromic inverted repeats Site-specific cleavage of duplex DNA Cleavage sites symmetrical Staggered or even cuts Prokaryotes 3-letter abbreviation of host, strain number if needed, and roman numeral Prokaryotes self DNA is not cleaved Recognition site methylated in self DNA Palindrome - A segment of double-stranded DNA in which the nucleotide sequence of one strand reads in reverse order to that of the complementary strand. Palindrome - A segment of double-stranded DNA in which the nucleotide sequence of one strand reads in reverse order to that of the complementary strand.

    6. Restriction Digest Mapping Separate restriction fragments by gel electrophoresis Resolving power 1 bases Polyacrylamide fragments less than 1000 bp Agarose fragments between 1 kb 20 kb PFGE agarose; pulse electric field - mb DNA bands visualized in gel by autoradiography or staining Radiolabel Fluorescent dye ethidium bromide, picrogreen

    8. Blotting Techniques Southern Blotting (DNA Blots) Transfer of DNA to a solid support for identification and characterization Northern Blotting (RNA Blots) Transfer of RNA to a solid support for identification and characterization Western Blotting (Protein Blots) Transfer of protein to a solid support for identification and characterization Probe with labeled antibody

    9. Southern Blotting Technique Restriction digest of duplex DNA Electrophoresis to separate fragments Denature duplex DNA to single stranded DNA Transfer ssDNA to nitrocellulose membrane Hybridize with labeled probe containing a complementary sequence Detection of hybridized probe

    11. DNA Sequencing De novo synthesis of DNA Sanger Dideoxy Method DNA polymerase for synthesis needs primer Controlled termination of replication 2,3-dideoxy analog of one of the nucleotides, different for each reaction mixture Four reaction mixtures containing labeled dNTPs Separation of four reactions using electrophoresis Base sequence read from autoradiogram (fluorescence scan) base read is complementary to 2,3-dideoxy analog

    14. Fluorescent tag attached to priming fragment; different color for each terminating analog Fluorescence tag can be added to dideoxy analogs and all four reactions completed in same tubeFluorescent tag attached to priming fragment; different color for each terminating analog Fluorescence tag can be added to dideoxy analogs and all four reactions completed in same tube

    16. Synthesis of DNA Automated Solid-Phase Methods Growing chain linked to an insoluble support Activated monomers sequentially added to growing chain The desired product remains on the solid support until the final release step Reactions take place in single vessel and excess reagents can be added to drive the reaction to completion After each step soluble reagents and by-products are easily removed from the immobilized growing chain

    17. Automated Solid-Phase Synthesis of DNA The 3-OH of a dNMP is immobilized on a glass bead. The amino groups on the bases are blocked An incoming activated monomer is joined to the 5-oxygen atom of the immobilized dNMP via the 3-OH

    19. Automated Solid-Phase Synthesis of DNA The 5-OH of the activated monomer is blocked with DMT (dimethoxytrityl) The 3-P of the activated monomer is blocked with ?CE (?-cyanoethyl group) Amino groups on the bases also blocked

    21. Automated Solid-Phase Synthesis of DNA Coupling of activated monomer to growing showing performed under anhydrous conditions to prevent the reaction of water with the phosphoramidites

    22. Automated Solid-Phase Synthesis of DNA Phosphite triester is oxidized by iodine The DMT protecting group on the 5-OH of the growing chain is removed by dichloroacetic acid Dichloroacetic acid leaves the other protecting groups intact

    24. Automated Solid-Phase Synthesis of DNA Each cycle takes about 10 min and elongates more than 98% of the chains At the end of synthesis, ammonia is used to remove all protecting groups and to release the oligonucleotide from the solid support The strands are of diverse length and can be purified using polyacrylamide gel electrophoresis or HPLC

    25. Applications Primers Label PCR Protein Engineering Tailor made genes Produce new proteins with novel properties in abundance

    26. Polymerase Chain Reaction Amplify specific DNA sequences Need to know flanking sequence of target Reaction Pair of primers that hybridize to the flanking sequences of the target dNTPs DNA polymerase heat stable

    27. Polymerase Chain Reactions Strand Separation 95oC for 15 s Hybridization of Primers 54oC Primer in excess Parent duplexes do not form DNA Synthesis 72oC Both strands 5 to 3 direction

    28. Polymerase Chain Reactions Target Sequences: 3-TAGCGTACG-5 5-ATCGCATCG-3 Primer Sequences: P1: 5-ATC P2: 5-CGA

    29. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGCATCG-3

    30. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATC AGC-5 5-ATCGCATCG-3

    31. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATC? ?AGC-5 5-ATCGCATCG-3

    32. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCG TAGC-5 5-ATCGCATCG-3

    33. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGC GTAGC-5 5-ATCGCATCG-3

    34. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGCA CGTAGC-5 5-ATCGCATCG-3

    35. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGCAT GCGTAGC-5 5-ATCGCATCG-3

    36. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGCATGC TAGCGTAGC-5 5-ATCGCATCG-3

    37. Polymerase Chain Reactions 3-TAGCGTACG-5 5-ATCGCATGCXXXXXXXXXXXXXXX XXXXXXTAGCGTAGC-5 5-ATCGCATCG-3

    44. Polymerase Chain Reactions Target sequence can be unknown as long as flanking sequence information is known Target sequence can be larger than primer 10 kb Primer sequence doesnt have to match exactly the flanking sequence PCR is highly specific Stringent conditions PCR is highly sensitive Amplify a single molecule

    45. PCR Application Diagnostic in medicine Detection of bacteria and viruses Forensic DNA profile Highly variable loci within a population Analysis of blood stains and semen samples Ancient DNA

    47. Recombinant DNA Technology New combinations of unrelated genes constructed in the lab Cloned Amplified Transcribed Translated

    48. Gene Construct Restriction enzyme digest of DNA fragment and plasmid DNA Insertion of DNA fragment into vector Cohesive Ends Decameric Linker DNA Ligase Vectors: Plasmids and BacteriophageVectors: Plasmids and Bacteriophage

    51. Cloning Using Vectors Vector Enhance the delivery of recombinant DNA into bacteria Select the bacteria containing the vector Plasmids Circular duplex DNA Occur naturally in some bacteria Bacteriophages Virus Circular single-stranded DNA

    52. Plasmids Genes for production of toxins, breakdown of natural products, inactivation of antibiotics Accessory chromosomes can replicate independently of host chromosomes pBR322 Genes for tetracycline and ampicillin resistance Eco RI site doesnt deactivate genes HindIII, SalI, BamHI sites deactivate gene for tetracycline resistance Selection of bacteria containing construct

    55. Bacteriophage Lambda (?) Phage Lytic Pathway Viral functions fully expressed Lysogenic Pathway Insert in host genome and replicated along with host genome Can remain inactive for years Environmental changes can trigger expression of dormant viral DNA Foreign DNA replaces a segment of ? phage DNA without affecting its function

    60. Bacteriophage M13 Phage Useful for sequencing inserted DNA Doesnt kill host Single-stranded DNA Replicative form is double-stranded but single-strand package into viral particle Sequencing Single-stranded DNA produced DNA Polymerase

    62. Artificial Chromosomes Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosomes (YACs) Autonomous replication sequence, a centromer, a pair of telomeres, selectable marker geners, and cloning site Restriction enzymes Separate fragments by pulsed-field gel electorphoresis Ligated to YACs

    64. Genomic Library Total genomic DNA Fragment genomic DNA Restriction enzymes Mechanically shearing Separate by size 15 kb Electrophoresis

    66. Genomic Library Gene Construct Synthetic Linkers added Fragments inserted into vector Cloning Infect bacteria with phage Phage Propagated indefinitely Genomic library Screening for gene of interest using DNA hybridization

    68. Complementary Probe mRNA from cells in which gene is available mRNA for hemoglobin in red blood cells Purify the mRNA cDNA synthesized in vitro cDNA cloned to produce probe Prepare from amino acid sequence of the protein encoded by the gene Obtain DNA sequence from amino acid sequence Met, Trp

    70. Designed Mutagensis Deletions Restriction digest of plasmid and ligate to form smaller circle Single cut, exonuclease treatment, and ligate to form circle

    71. Designed Mutagensis Substitutions Point Mutation Prepare prime with mutation; use to synthesize DNA

    73. Designed Mutagensis Insertions Synthetic double-stranded oligonucleotide with desired mutations ligated into plasmid

    75. Designer Genes Create novels proteins Splicing different gene segments Synthetic gene via solid-phase method

    77. Manipulating Genes Bacteria can be used to produce a protein product Eukaryotic DNA has introns and exons Prokaryotic DNA does not have introns and exons

    78. Problem How can a prokaryotic system process eukaryotic DNA????

    79. Obstacle They Can Not!!!!!!!!!!!!!!!!

    80. Solution cDNA What is cDNA?

    81. cDNA Complementary DNA DNA is complementary to mRNA mRNA ? cDNA Reverse Transcriptase mRNA has poly (A) tail; make a DNA primer containing oligo(T) residues RNA-DNA hybrid; raise pH to hydrolyze RNA ssDNA ? dsDNA Terminal transferase adds dG to 3 end and oligo (dC) as primer

    84. Alkali Digestion of mRNA

    89. Metallothionein activated by addition of heavy metalsMetallothionein activated by addition of heavy metals

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