U-M SI 501 - Recombinant DNA Technology (27 pages)

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Recombinant DNA Technology



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Recombinant DNA Technology

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Pages:
27
School:
University of Michigan
Course:
Si 501 - Contextual Inq
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Recombinant DNA Technology Stephen B Gruber MD PhD Division of Molecular Medicine and Genetics November 4 2002 Learning Objectives Know the basics of gene structure function and regulation Be familiar with the basic methods of molecular genetics Understand the meaning of DNA sequence and amino acid polymorphisms Know how DNA sequence analysis is performed and be familiar with methods of screening for differences Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies 1 Learning Objectives 1 Know the basics of gene structure function and regulation Be familiar with the basic methods of molecular genetics Understand the meaning of DNA sequence and amino acid polymorphisms Know how DNA sequence analysis is performed and be familiar with methods of screening for differences Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies Chromosomes DNA and Genes Gene Cell Nucleus Chromosomes Protein Adapted from Understanding Gene Testing NIH 1995 2 Genetic Code A codon is made of 3 base pairs 64 codons total 1 codon AUG encodes methionine and starts translation of all proteins 61 codons encode 20 amino acids redundant code 3 codons stop protein translation A U G G C A U A A Met Ala DNA Transcription and Translation Growing chain of amino acids mRNA Ribosome DNA Nuclear membrane Protein Cell membrane Adapted from Understanding Gene Testing NIH 1995 3 Gene Structure RNA transcription start site Splice sites Stop site Promoter Exon 1 Intron Exon 2 Intron Exon 3 5 end 3 end Exon 1 Exon 2 Exon 3 mRNA RNA Processing Exon Intron Exon Intron Exon DNA Transcription Primary mRNA GU AG Processing Mature mRNA Translation Protein 4 Learning Objectives 2 Know the basics of gene structure function and regulation Be familiar with the basic methods of molecular genetics nucleic acid hybridization Southern DNA and northern RNA blotting PCR DNA sequencing basic steps involved in constructing screening a cDNA library Understand the meaning of DNA sequence and amino acid polymorphisms DNA sequence analysis Transgenic technologies 1983 Huntington Disease gene mapped 1981 Transgenic mice 1956 Glu 6 Val in sickle hemoglobin 1944 DNA is the genetic material 1945 1970 First restriction enzyme 1953 Double helix 1950 1949 Abnl Hemoglobin in sickle cell anemia 1955 1960 1965 1966 Completion of the genetic code 1970 1975 Southern blotting 1975 1972 Recombinant plasmids 1980 2001 Draft human genome sequence 1989 Positional cloning without deletion CF 1995 1st complete bacterial genome sequence 1985 PCR 1985 1986 Positional cloning CGD muscular dystrophy retinoblastoma 1990 1995 2000 1996 1990 Complete yeast First NIHgenome sequence approved gene therapy experiment 1987 Knockout mice from Textbook 5 4 5 Preparing DNA for Analysis DNA for analysis Centrifuge and extract DNA from white blood cells Blood sample SINGLE STRANDED DNA PROBES FOR GENE A C D B MIXTURE OF SINGLE STRANDED DNA MOLECULES E A F C C D B B D A A E E F ONLY A FORMS A STABLE DOUBLE STRANDED COMPLEXES STRINGENT HYBRIDIZATION F A C E ALL FORM STABLE COMPLEXES REDUCED STRINGENCY HYBRIDIZATION Textbook Figure 5 8 6 Electrophoresis of DNA DNA fragments loaded into wells DNA fragments separate by size and charge Voltage Path of migration Principle of a Southern blot hybridize labeled probe to fragment of DNA Electrophoresis Restriction enzyme digestion Add radio labeled normal DNA probes 7 Polymerase Chain Reaction PCR Isolate and denature DNA Anneal and extend primers Repeat as necessary Amplified segments Sequence to be amplified DNA Sequencing 8 SINGLE STRANDED DNA OF UNKNOWN SEQUENCE 5 C T G A C T T C G A C A A 3 RADIOACTIVELY LABELED 3 T G T T PRIMER DNA POLYMERASE I dATP dGTP dCTP dTTP ddATP ddCTP ddTTP P P P O 5 CH 2 O BASE H H H H H H DIDEOXYNUCLEOTIDE ddNTP ddGTP REACTION MIXTURES C T G A C T T C G A C A A GEL ELECTROPHORESIS ddG ddG ddGTP ddTTP ddCTP ddATP AUTORADIOGRAPHY TO DETECT RADIOACTIVE BANDS ddG PRODUCTS IN ddGTP REACTION C T G LARGER FRAGMENTS READ SEQUENCE OF ORIGINAL SINGLE STRANDED DNA COMPLEMENT OF PRIMERGENERATED SEQUENCE LADDER A C T T C G SMALLER FRAGMENTS Textbook Figure 5 17 DNA Sequencing AG ATC TTA GTG TCC C ATC TTA GAG TGT CCC A T C G A T C delG delA Start Normal Start G Mutant 185delAG 9 Learning Objectives 3 Know the basics of gene structure function and regulation Be familiar with the basic methods of molecular genetics nucleic acid hybridization Southern DNA and northern RNA blotting PCR and gel electrophoresis DNA sequencing basic steps involved in constructing screening a cDNA library Understand the meaning of DNA sequence and amino acid polymorphisms DNA sequence analysis Transgenic technologies Polymorphisms and Mutations Sequence variation differences among individuals DNA amino acid 0 01 polymorphism 0 01 rare variant Mutation any change in DNA sequence Silent vs amino acid substitution vs other neutral vs disease causing Common but incorrect usage mutation vs polymorphism balanced polymorphism disease polymorphism 10 Learning Objectives 3 continued Understand the meaning and significance of DNA sequence and amino acid polymorphisms Understand the various types of DNA sequence polymorphisms RFLPs Restriction Fragment Length Polymorphism VNTRs Variable Number Tandem Repeat SSRs Simple Sequence Repeat also STR Short Simple SNPs Tandem Repeat Single Nucleotide Polymorphism Textbook Figure 5 19 11 Learning Objectives 3 continued Understand the meaning and significance of DNA sequence and amino acid polymorphisms Understand the various types of DNA sequence polymorphisms RFLPs Restriction Fragment Length Polymorphism VNTRs Variable Number Tandem Repeat SSRs Simple Sequence Repeat also STR Short Simple SNPs Tandem Repeat Single Nucleotide Polymorphism Disease Associated Mutations Alter Protein Function Functional protein Nonfunctional or missing protein 12 P1 P2 TCTA 10 A TCTA 11 B TCTA 12 C TCTA 13 D TCTA 14 E TCTA 15 F 15 14 13 12 11 10 AB CD EF AF CE Textbook Figure 5 22 SNP coding sequence Normal mRNA Protein Sequence variant A U G A A G U U U G G C G C A U U G C A A Met Lys Phe Gly Ala Leu Gln A U G A A G U U U G G U G C A U U G C A A Met Lys Phe Gly Ala Leu Gln mRNA Protein Silent DNA sequence polymorphism 13 Disease Associated Mutations A mutation is a change in the normal base pair sequence Commonly used to define DNA sequence changes that alter protein function Polymorphism DNA


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