11/19/2008. Gene analysis. Sequencing PCR. Northern-blot RT PCR. Western-blot Sequencing. in situ hybridization. Southern-blot

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1 Recombinant technology Gene analysis Sequencing PCR RNA Northern-blot RT PCR Protein Western-blot Sequencing Southern-blot in situ hybridization in situ hybridization Function analysis Histochemical analysis Analysis of nucleic acids analysis: Identification of gene mutations precise diagnosis of genetic diseases (prenatal, postnatal); Identification of individual polymorphism genomic fingerprint (analysis of filiations); Identification of foreign (bacterian, viral) diagnostic of infectious diseases and level of infection. Analysis of mrna: Tissue-specific gene expression; Identification of level of gene expression (for normal and pathological genes). 1

2 isolation and purification Collecting of biological material (nuclear cells from all tissues; bacterial cells, fluids containing viruses) Cell lysis in hypotonic solution Cell lysis with detergents Deproteinisation: proteatsis and organic solvents Precipitation of nucleic acids with alcohol RNA isolation and purification Collecting of biological material (interested tissue, at interested step of development and conditions) Cell lysis with detergents in presence of inhibitors of RN-ases Deproteinisation with organic solvents Selective precipitation of RNAs with salts (LiCl, CH 3 COONa) Precipitation of RNAwith alcohol Human Gene to be cloned Cutting with enzyme Plasmid cloning vector Ligation Recombinant a molecule containing sequences from different sources Host cell Recombinant Genetic transformation Cloning Expression of human gene 2

3 Objectives of recombinant technology Cloning of interested sequence / gene for: multiplication; Construction of libraries; Molecular analysis. Gene expression for: Functional analysis of gene; Preparation of protein. Steps in recombinant preparation 1. Isolation of interested and cloning vectors 2. Enzymatic cleavage of interested and cloning vectors 3. Ligation of interested fragment of and vector recombinant 4. Introduction of recombinant molecules into host cell genetic transformation 5. Cloning of interested fragment by in vivo replication 6. Expression of interested gene by protein synthesis 7. Screening of transformed cells using antibiotics and radioactive / fluorescent markers Components required for cloning in vivo - to be cloned: restriction fragments, c - Cloning vectors: - molecules able to survive in foreign cell - able to transport foreign fragments of - able to replicate independently - Restriction enzymes - Cloning host cell, compatible with vector - A system for screening of clones - Enzymes for preparation 3

4 The length of cloned in different vectors Plasmids Bacteriophages Vector The length of fragment Host cell Up to 10 kb (genomic, c) Up to 25 kb (genomic, c) Bacteria, Yeast Bacteria Cosmids kb (genomic ) Bacteria BAC (Bacterial artificial chromosome) YAC (Yeast artificial chromosome) Up to 300 kb (genomic ) Bacteria 0,2-2.0 Mb (Genomic ) Yeast Vector: -Contains ori point (is able to replicate independently) -May carry foreign fragments (contains restriction sites) -Contains genes that ensure resistance against antibiotics Bacterial cell Ligation of insertions with vector 4

5 Preparation of Recombinant to be cloned - cloning vector Identification of RS BamH I Restriction site RS BamH I RS BamH I Required fragment Hydrolysation with restrictase RF RF RF RF RF Ligation Recombinant Obtaining of restriction fragments using restriction enzymes Identification of RS RS BamH I RS Hae III RS BamH I RS Hae III Hydrolysation with RE BamH I Hae III Restriction fragments (RF) RF 1 Sticky ends RF RF 2 Sticky ends 3 Sites recognized by different restriction enzymes 5

6 A. isolated from patient X RS Hae III RS Hae III Identification of RS Hydrolysation with RE RF 1X RF 2X RF 3X B. isolated from patient Z RS Hae III Identification of RS Hydrolysation with RE RF 1Z RF 2Z RFLPs Restriction Fragments Length Polymorphism of patients X and Z RE restriction enzyme site specific endonuclease that recognizes and cuts double-stranded RS restriction site a palindrome sequence recognised by RE RF restriction fragment a fragment of doublestranded obtained after restriction of RFLPs Restriction Fragments Length Polymorphisms individual differences between molecules Restriction map a linear physical map of a region of, drawn to show the relative positions of the target sites of various restriction enzymes - 12 kb 10 kb 10 kb 8 kb 2,5 kb 10 kb 6 kb 5 kb 6 kb 5 kb 2,5 kb 6 kb 4 kb 2 kb + M 6

7 Human Gene to be cloned Host cell Cutting with enzyme Ligation Plasmid cloning vector Recombinant Genetic transformation in vivo cloning of a human gene Problem Human genes are large; Regulatory regions differ from bacterial; Contains introns, that cannot be removed in bacterial cells. Cloning Expression of human gene Solution: Synthesis of c on the basis of mrna corresponding to target gene 7

8 Steps in c synthesis (complementary ) Isolation of Poly(A) + RNA Hybridization of poly(a) with oligo(dt) Synthesis of first strand of c using reversetranscriptase Hydrolyzation of RNA Synthesis of the second strand using -polymerase Visualization of Using specific dies Using labeled probes Basic Fuxin (in situ) Fluorescent (cold) Radioactive (hot) Ethidium bromide (in gels) Large amounts of required Is necessary to know the sequence of studied Practical role of analysis of : 1. Research sequencing of, construction of gene libraries, analysis of gene expression, analysis of regulatory mechanisms, analysis of mutations. 2. Identification of mutation carriers prenatal or postnatal, prevention of birth of children with anomalies or strong defects. 3. Gene therapy (introduction of normal gene in a carrier of mutation). 4. Identification of foreign (viral or bacterial) in diagnosis of infectious diseases. 5. Identification of individual polymorphisms in filiations, criminology. 8

9 cloning = multiplication of by repetitive replication in vivo Recombinant Host cell required Replication using a vector which contains a ori point Possibility to obtain final product - protein PCR in vitro In artificial conditions Replication using an artificial system in vitro cloning PCR (polymerase chain reaction) Components required for in vitro cloning (PCR): - to by analyzed - Specific primers to initiate replication - Taq-polymerase - dntp - Termocycler: - tº for denaturation of - tº for primer attachment - tº for polymerization (elongation of ) 9

10 Steps of PCR A. Preparation of mixture for reaction B. Denaturation renaturation elongation cycles Denaturation of at 96 o C Cooling up to renaturation temperature Elongation at 72 o C C. Analysis of amplified products - electrophoresis - staining - interpretation of results Gene libraries Genomic libraries contain sequences of genomic from organism c libraries contain c sequences obtained on the basis of mrna from a tissue Chromosomal libraries contains sequences specific to a chromosome 10

11 Steps in preparation of genomic libraries Isolation of genomic Restriction of genomic Ligation of restriction fragments with vector Genetic transformation Clone selection Steps in preparation of c libraries Isolation of mrna Synthesis of c Ligation of linkers to ends of c Restriction of c and vector Ligation cvector Genetic transformation Clone selection 11

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