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Unformatted text preview: 204-325 1 Summary of the Last lecture Using restriction enzymes and DNA ligase, a recombinant DNA molecule containing an insertion into a vector (plasmid, BAC or YAC) can be made in a test tube. By making genomic or cDNA library, a gene of interest can be isolated by screening the library with antibody or DNA probe. Restriction mapping is the determination of locations of restriction sites on a DNA fragment. Complete DNA sequence can be determined by using dideoxyribonucleotides and electrophoresis. Polymerase chain reaction (PCR) can amplify a specific DNA segment bracketed by two primers
2 Today's Topics 1. DNA Polymorphism: Detection and Application 2. Positional Cloning 3. Genetically Modified Organisms 4. Gene Therapy 3 Topic 1 DNA Polymorphism:
Detection and Application
4 DNA Polymorphisms in Human Genome as Markers 1.Single Nucleotide Polymorphism (SNP): 90% of all human genetic variation, occurs every 100 to 300 bases.
RFLP if a resection site is altered More general - ASO 2. Short Tandem Repeat (STR, SSR, microsatellite): RFLP or PCR 3. Variable Number of Tandem Repeat (VNTR, minisatellite)
5 Southern Blot 6 Detecting SNP that Alters a Restriction Site by Southern Blot Codominant 7 SNP in -globin Gene: Normal (Hb-A) and Mutant (Hb-S) Forms DdeI 8 Restriction Fragment Length Polymorphism (RFLP) 9 Detecting SNP that Alters a Restriction Site by PCR-RFLP 10 SNP Detention by Allele-Specific Oligonucleotide (ASO) Hybridization GLC1A (normal/glaucoma) 11 Animation 1: DNA Testing for Human Disease Genes 12 Repetitive DNA Sequences within a Genome
Interspersed Repeated DNA Short Interspersed Sequence (SINE): 100-500 bp Long Interspersed Sequence (LINE): 5000 bp or more Tandemly Repeated DNA Short Tandem Repeat (STR): 2-4 bp (microsatellite) Variable Number of Tandem Repeat (VNTR): 5-100 bp (minisatellite)
Human Genome 13 Examples of VNTR 14 Detection of VNTR Polymorphism by RFLP 15 DNA Fingerprinting by RFLP RFLP: Restriction Fragment Length Polymorphism 16 Detection of STR Polymorphism by PCR PCR with primers X and Y and analyze by gel electrophoresis EtBr stained gel =PCR product size 17 DNA Fingerprinting by PCR 18 5264 STRs in Human genome 19 13 FBI CODIS STR loci as "Personal Bar Code"
Combined DNA Index System (CODIS ) 20 How to Read the "Personal Bar Code"?
FAM (Blue) JOE (Green) TAMRA (Yellow) ROX (Red) TH01 D7 D5 VWA D13 D21 D8 TPOX CSF D16 D18 FGA Penta D Penta E D3 AMEL PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis
21 Forensic Application: FBI CODIS STR loci
Combined DNA Index System (CODIS ) 22 Application: Paternity Determination 23 Topic 2 Positional Cloning
24 Positional Cloning of Human Disease Gene Overview Cytogentic Abnormality Chromosome Walking Physical Mapping & Cloning Transcript Identification Family Finer Genetic Mapping Mutation Identification Genetic Markers YAC BAC Positional Candidate 25 Chromosome Walking 26 Chromosome Walking 27 Chromosome Walking 28 Positional Cloning of CF Gene Normal DNA ... GAA AAT ATC ATC TTT GGT GTT... Protein Glu Asn Ile Ile Phe Gly Val Position 504 505 506 507 508 509 510 DNA ... GAA AAT ATC ATT GGT GTT... Protein Glu Asn Ile Ile Gly Val
29 Cystic Fibrosis F508 Topic 3 Genetically Modified Organisms
30 Infection of Plant Roots by Agrobacterium 31 Making a Transgenic Roundup-tolerant Plant 32 Animation 2: Plant Genetic Engineering 33 Golden Rice Containing -carotene to Make Vitamin A 34 Introducing Foreign Gene into Animal Chromosome 35 Steps in Making Transgenic Mouse 36 Production of Desirable Proteins in Transgenic Sheep 37 Transgenic Salmon with Growth Hormone (1994) 38 GloFish: Transgenic Zebrafish (2004) 39 Topic 4 Gene Therapy
40 Germinal versus Somatic Gene Therapy 41 In Vivo Gene Therapy
stable gene expression, but may cause mutations, gene size limit no gene size limit, but low efficiency no gene size limit, but only for dermal tissue 42 Ex Vivo Gene Therapy
DNA Liposome Plasmid 43 An Example of Ex Vivo Gene Therapy 44 Summary RFLP, PCR and ASO hybridization are used to detect DNA polymorphisms. Almost all human beings are genetically distinct. SNP and the variable length of STRs or VNTRs can be used to identify an individual, detect paternity and for forensic applications. Genes can be cloned based on their genetic linkages with markers. Positional cloning is a powerful tool for identifying human disease genes. We are able to genetically modify or engineer many organisms for medical, agricultural and other purposes. Human gene therapy is limited to somatic tissues at present.
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