Sequencing and Microarray Analysis PDF

Sequencing and microarray analysis 2 5. Sequencing of gene expression  The previouslystudied DNA sequencing techniques were first developed for DNA and genome analysis.  Gene identification has grown rapidly and complete sequencing of genomes was reached.  Once the gene is found an...

Sequencing and microarray analysis 2 5. Sequencing of gene expression  The previouslystudied DNA sequencing techniques were first developed for DNA and genome analysis.  Gene identification has grown rapidly and complete sequencing of genomes was reached.  Once the gene is found and isolated, experiments can be done to study the expression of the gene in normal and mutant organisms.  Allows the understanding of the role of the gene in determining the phenotype and its cellular functions.  New lines of research became possible, such as the analysis of expression of all genes in a cell at the transcriptional and translational level.  Measuring the levels of mRNA transcripts gives us insight into the global gene expression state of the cell Transcriptome  Sequencing and microarray analysis 3 5. Sequencing of gene expression The Transcriptome  The transcriptome is a major indicator of cellular phenotype and function.  The transcriptome is not the same in all the cells in an organism: 2 different cell types will transcribe overlapping but very different subsets of the genes in the genome. The same cell type will have a constant transcriptome but may change if the cell changes (growth, differentiation…).  4 5. Sequencing of gene expressionSequencing and microarray analysis The Transcriptome Normal cell Cancerous cell 209 1327 134 Genes exclusively Genes exclusively expressed in expressed in normal cells cancerous cells Genes with higher Genes with higher expression in Genes equally expression in normal cells expressed = cancerous cells housekeeping genes  Sequencing and microarray analysis 5 5. Sequencing of gene expression The Transcriptome  The transcriptome is a major indicator of cellular phenotype and function.  The transcriptome is not the same in all the cells in an organism: 2 different cell types will transcribe overlapping but very different subsets of the genes in the genome. The same cell type will have a constant transcriptome but may change if the cell changes (growth, differentiation…).  Analyzing the transcriptome is important to understand cellular function at a global level and the entirety of the cellular response to a particular condition.  Transcriptome studies use DNA microarrays (also called gene chips or DNA chips) to investigate global gene expression.  5. Sequencing of gene expression Sequencing and microarray analysis 6 DNAmicroarrays  Another technique based on the concept of nucleic acid hybridization.  Monitoring the expression of thousands of genes simultaneously.  A DNA microarray consists of an organized array of thousands of individual, closely packed, gene-specific sequences attached to the surface of a glass microscope slide (DNA Chip).  5. Sequencing of gene expression Sequencing and microarray analysis 7 DNAmicroarrays  Sequencing and microarray analysis 8 5. Sequencing of gene expression DNAmicroarrays  Another technique based on the concept of nucleic acid hybridization.  Monitoring the expression of thousands of genes simultaneously.  A DNA microarray consists of an organized array of thousands of individual, closely packed, gene-specific sequences attached to the surface of a glass microscope slide (DNA Chip).  By coupling microarray analysis with the results from genomic sequencing projects, we can analyze the global patterns of gene expression in an organism during specific physiological responses or developmental processes.  Sequencing and microarray analysis 9 5. Sequencing of gene expression DNAmicroarrays procedure 1) The initial step in a microarray expression study is to prepare cDNAs from the mRNAs expressed by the cells under study 2) attach fluorescent labels to them (Cy3 or Cy5). 3) When the cDNA preparation is applied to a microarray under appropriate conditions, DNA spots representing genes that are expressed will hybridize to their complementary cDNAs 4) labeled probes can subsequently be detected in a scanning laser microscope.  5. Sequencing of gene expression Sequencing and microarray analysis 10 DNAmicroarrays procedure  5. Sequencing of gene expression Sequencing and microarray analysis 11 DNAmicroarrays procedure  5. Sequencing of gene expression Sequencing and microarray analysis 12 DNAmicroarrays results  Sequencing and microarray analysis 13 5. Sequencing of gene expression DNAmicroarrays results A micrograph of a small segment of an actual DNA microarray. Each spot in this 16 × 16 array contains DNA from a different gene hybridized to control and experimental cDNA samples labeled with red and green fluorescent dyes. (A yellow spot indicates equal hybridization of green and red f luores cence, indicating no change in gene expression.) Microarray analysis of gene expression in fibroblasts showed that transcription of about 500 of the 8600 genes examined changed substantially after addition of serum  5. Sequencing of gene expression Sequencing and microarray analysis 14 ClusterAnalysis of multiple expression  Genes that exhibit similar changes in expression cannot be termed co- regulated from a single microarray experiment.  Combine the information from a set of microarray expression experiments to find genes that are similarly regulated under a variety of conditions or over a period of time = Clustering analysis.  Cluster analysis groups sets of genes whose encoded proteins participate in a common cellular process.  Sequencing and microarray analysis 15 5. Sequencing of gene expression ClusterAnalysis of multiple expression Five clusters of coordinately regulated genes were identified in this experiment, as indicated by the bars at the bottom. Each cluster contains multiple genes whose encoded proteins function in a particular cellular process: cholesterol biosynthesis (A), the cell cycle (B), the immediate early response (C), signaling and angiogenesis (D), and wound healing and tissue remodeling (E).  Sequencing and microarray analysis 16 6. Next generation sequencing (NGS)  NGS also known as high-throughput sequencing, is used to describe a number of different modern sequencing technologies.  These technologies allow for sequencing of DNA and RNA much faster and cheaper than the previously used Sanger sequencing.  Some of these technologies emerged in 1990’s and have only been commercially available since 2005.  These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads (50-400 bases each) per instrument run.  Different sequencing methods : Bridge sequencing SMRT sequencing  Sequencing and microarray analysis 17 6.a. Bridge sequencing (Illumina)  In Illumina sequencing, 100-150bp reads (DNA sequences) are used.  Genomic DNA is first fragmented. Or RNA is retrotranscribed to cDNA fragments.  These fragments (gDNA or cDNA) are ligated to generic adaptors and annealed to a slide using the adaptors.  PCR is carried out to amplify each read, creating a spot with many copies of the same read.  They are then separated into single strands to be sequenced.  This technique is also called Sequencing By Synthesis (SBS).  Sequencing and microarray analysis 18 6.a. Bridge sequencing (Illumina)  Sequencing and microarray analysis 19 6.a. Bridge sequencing (Illumina)  Sequencing and microarray analysis 20 6.a. Bridge sequencing (Illumina) Cluster 10-200 million/flow cell Each cluster generates a single read  At the end, Flow cell 10-200 million sequenced DNA fragments  Sequencing and microarray analysis 21 6.a.BridgeAmplification  Sequencing and microarray analysis 22 6.a.Bridge sequencing (Illumina)  Sequencing and microarray analysis 23 6.a.Bridge sequencing (Illumina) Each Cluster is read separately and bases are called depending on fluorescence emitted Cluster  Sequencing and microarray analysis 24 6.b. Single Molecule real time (SMRT) sequencing  Third generation sequencing is developed with this technique.  Allows sequencing of long reads >10kb with very high accuracy.  No PCR amplification step.  Uses a zero-mode waveguide chamber to analyze single molecule addition to DNA. A structure designed to allow observation of a single base as it is being incorporated by the polymerase  Sequencing and microarray analysis 25 6.b. Single Molecule real time (SMRT) sequencing Zero-mode waveguide chamber contains one DNA molecule Labeled dNTPs Fixed DNA Pol  Sequencing and microarray analysis 26 6.b. Single Molecule real time (SMRT) sequencing  Third generation sequencing is developed with this technique.  Allows sequencing of long reads >10kb with very high accuracy.  No PCR amplification step.  Uses a zero-mode waveguide chamber to analyze single molecule addition to DNA.  Each of the four DNA bases is assigned a different fluorescent dye, so as each new base is incorporated, it gives off a distinct, identifiable signal.  After incorporation, the fluorescent tag is removed and the next base is added.  Sequencing and microarray analysis 27 6.b.Single Molecule real time (SMRT) sequencing  Sequencing and microarray analysis 28 6.b.Single Molecule real time (SMRT) sequencing  Sequencing and microarray analysis 29 6.b.Single Molecule real time (SMRT) sequencing  Sequencing and microarray analysis 30 6.Next Generation Sequencing  Produces huge amounts of data (up to 2 Tbytes/ run) to be analyzed (bioinformatics in a later topic)  Has many advantages: Higher sensitivity to detect low-frequency variants Faster turnaround time for high sample volumes Comprehensive genomic coverage Lower limit of detection Higher throughput with sample multiplexing Ability to sequence hundreds to thousands of genes or gene regions simultaneously  Fourth and Fifth generation of NGS are developed to improve speed and accuracy of sequencing.  Gene mapping 1 MLAB 230 - Molecular Biology Topic 8: Gene mapping  Gene mapping 2 Gene mapping  Define the positions of genes on a DNA molecule or chromosome and the distance between them.  Based on different genetic and molecular techniques and the use of several genetic markers (SNP, STR…).  2 types of maps can be obtained : Genetic map : based on the use of genetic techniques to construct maps showing the location of genes (cross-breeding, pedigree…) Physical map : uses molecular biology techniques to examine DNA molecules directly to construct maps showing the position of feature sequences (genes, promoters, consensus and conserved sequences…)  Mapping a gene is the first step of identification and the starting point of analyzing disease manifestation in large families or from populations-based genetic association studies (GWAS).  Gene mapping 3 Mendelian laws: an overview  Mendel’s work led to the definition of 3 laws : 1. The law of dominance : a dominant trait is a trait whose appearance will always be seen in offspring. If an individual inherits two different alleles from each of its two parents and the phenotype of only one allele is visible in the offspring, then that allele is said to be dominant. 2. The law of segregation : If a parent has two distinct alleles for a certain gene, each on one copy of a given chromosome, these two alleles will be separated from each other during meiosis. 3. The law of independent assortment : the way an allele pair gets segregated into two daughter cells during the second division of meiosis has no effect on how any other allele pair gets segregated. The traits inherited through one gene will be inherited independently of the traits inherited through another gene because the genes reside on different chromosomes  Gene mapping 4 Mendelian laws: an overview Law of Dominance  Gene mapping 5 Mendelian laws: an overview Law of Segregation  Gene mapping 6 Mendelian laws: an overview Law of Independent Assortment 50% 50% 25% F2 generation : 25% 25% 3/4 Dominant phenotype R 1/4 Recessive phenotype r 25%  Gene mapping 7 Mendelian laws:Applied on 2 traits (color, shape) F2 generation : 9/16 phenotype AB 3/16 phenotype Ab 3/16 phenotype aB 1/16 phenotype ab  Gene mapping 8 The Thomas Hunt Morgan Experiment  Gene mapping 9 The Thomas Hunt Morgan Experiment Results : 965 944 206 185 Are Mendel’s laws F2 generation : as per mendel’s independent assortment law wrong? 1/4 phenotype b+ vg+ Parental phenotypes 50% 1/4 phenotype b vg 1/4 phenotype b+ vg Recombinant phenotypes 50% 1/4 phenotype b vg+  Gene mapping 10 The Thomas Hunt Morgan Experiment  If the 2 genes where on the same chromosome : F2 generation : as per mendel’s independent assortment law Only Parental 1/2 phenotype b+ vg+ phenotypes 1/2 phenotype b vg  Gene mapping 11 The Thomas Hunt Morgan Experiment  How can this result be explained? Results : 965 944 206 185  Gene mapping 12 The Thomas Hunt Morgan Experiment  Morgan’s group analyzed a large number of other crosses of this type. In each case : the parental phenotypic classes were the most frequent the recombinant classes occurred less frequently  Approximately equal numbers of each of the two parental classes, and approximately equal numbers of each of the two recombinant classes, were obtained  Morgan concluded that when 2 genes are on the same chromosome, they do not segregate independently. These are linked genes  the closer two genes are on the chromosome, the more likely they are to remain together during prophase I of meiosis.  the recombinants are produced as a result of crossing-over between homologous chromosomes during meiosis, and the closer two genes are together, the less likely there will be a recombination event between them.  Gene mapping 13 Genetic Cross-over  Gene mapping 14 Gene Linkage  The data obtained by Morgan from Drosophila crosses indicated that the frequency of crossing-over (and hence of recombinants) for linked genes is specific of the gene pairs involved.  In 1913, a student of Morgan’s, Alfred Sturtevant, determined that recombination frequencies could be used as a quantitative measure of the genetic distance between two genes on a genetic map.  The genetic distance between genes is measured in map units (mu).  1 map unit (mu) is defined as the interval in which 1 percent crossing- over takes place.  The map unit is also called a centimorgan (cM) 1mu = 1 cM  Gene mapping 15 Gene Linkage Results : 965 944 206 185 Total 2300 Recombinant frequency = (206 + 185) / 2300 = 0.17 or 17% The distance between both genes, body color and wing size, is 17 cM  Gene mapping 16 How to map a chromosome  If we add another gene to the cross-over analysis : Genes Recombination frequency Vg-b 17% Vg-cn 8% Cn-b 9%  The distance between Vg and cn is 8 cM  The distance between cn and b is 9 cM 17cM 8cM 9cM vg cn b  Gene mapping 19 Human genome mapping  Humans do not do controlled mating for obvious ethical reasons.  Humans have few offsprings.  Only about 2% of the human genome consists of genes, which means that genes are generally scattered widely in the genome constructing a human genetic map based on genes is an impossible task.  The discovery of DNA polymorphic markers (SNP…) made the mapping of the human genome possible.  DNA markers provide alleles of a locus that differ in a molecular phenotype, rather than a visible or biochemical phenotype while being more frequent in the human genome than are genes.  Gene mapping 20 Human genome mapping procedure  The alleles of polymorphic DNA marker loci are analyzed in DNA samples isolated from individuals in a large number of pedigrees.  Linkage between DNA marker loci is typically determined by the “lod score” (logarithm of odds) method using computer algorithms.  The lod score method compares: 1. the probability of obtaining the pedigree results if two genetic markers are linked with a certain amount of recombination between them 2. the probability that the results would have been obtained if there was no linkage (i.e., 50% recombination) between the markers  Once linkage is established between genetic markers, the map distance is computed from the recombination frequency giving the highest lod score (the higher the lod score the closer the genes are).  The human genome project and the development of new sequencing techniques have highly improved the mapping of the human genome.  Gene mapping 21 Human genome mapping procedure The diagram depicts a human chromosome analyzed at different levels of detail: The chromosome as a whole can be viewed in the light microscope and the approximate location of specific sequences can be determined by FISH. genetic traits can be mapped relative to DNA-based genetic markers. Local segments of the chromosome can be analyzed at the level of DNA sequences identified by Southern blotting or PCR. Important genetic differences can be most precisely defined by differences in the nucleotide sequence of the Chromosomal DNA  Gene mapping 22 Human disease genes  Most cases of inherited diseases are caused by preexisting mutant alleles that have been passed from one generation to the next for many generations.  The typical first step in deciphering the underlying cause of any inherited human disease is to identify the affected gene and its encoded protein  They must be found without any prior knowledge or reasonable hypotheses about the nature of the affected gene or its encoded protein  The segregation of the disease can be correlated with the segregation of many other genetic markers, eventually leading to identification of the chromosomal position of the affected gene.  Gene mapping 23 Genome WideAssociation Studies (GWAS)  GWAS are a relatively new way for scientists to identify genes involved in human disease.  This method searches the genome for small variations, SNPs, that occur more frequently in people with a particular disease than in people without the disease.  Each study can look at hundreds or thousands of SNPs at the same time.  Their purpose is to determine alleles that correlate to different diseases and traits.  GWAS is simple and begins by dividing participants into two groups: People with a disease/trait of interest People without a disease/trait (control group)  Gene mapping 24 Genome WideAssociation Studies (GWAS)  The SNPs of both groups are analyzed and compared with the hope of identifying a particular SNP(s) present in the disease group that is not present in the control group.  If such a SNP or SNPs are located then a genetic sequence that has an “association” with a particular disease has been identified.  Thousands of diseases and traits have been investigated by GWAS including: height, type 2 diabetes, pigmentation, epilepsy, body mass index, Alzheimer’s disease, autism…  Gene mapping 25 Genome WideAssociation Studies (GWAS)  Gene mapping 26 Genome WideAssociation Studies (GWAS) Analyzed by microarray or sequencing Manhattan plot of GWAS results  Gene mapping 27 Genome WideAssociation Studies (GWAS)  In GWAS it is important to recognize that the relationship of identified SNPs to a particular disease is only an association  GWAS analyze common variants (SNP arrays), they do not generally identify causal (gene) variants but denote regions containing those causal agents  Additional studies are usually required to narrow the region of association and identify the gene responsible for this association.  the results of GWAS are presented as an odds ratio, which is “a measure of the odds of an event happening in one group compared to the odds of the same event happening in another group”.  A percentage increased risk of the disease to an individual carrying a risk variant can then be calculated.  Gene mapping 28 Genome WideAssociation Studies (GWAS) GWAS of Basal cell carcinoma analysis included 12,945 cases and 274,252 controls Loci with smallest Pe.g Dolly the sheep Identical copies of gene => gene-cloning We will discuss in the following some of the gene-cloning  Gene Cloning strategies Libraries (genomic and cDNA) Gene isolation PCR Chemical synthesis Types of vectors Insertion into a Choice of vectors cloning vector Choice of cloning technique (use of restrict Transformation methods Introduction into host Transduction Transfection Introduction of selection genes Hybridization techniques Selection or screening PCR Immunochemical methods let us take an example You want to int bacteria or mam produce insulin - You need the Source? - You need a v what type? - You need to DNA (vector - You need to host. How? - You need to cells who po those who do  Gene Cloning strategies Libraries (genomic an Gene isolation PCR Chemical synthesis  Vectors: Definition What is a vector?  Vectors function as DNA carriers to the cell to allow 1. replication of recombinant DNAs Foreign DNA has no origin of replication 2. selection of cells who incorporated the recombina 3. If required, promoter and other gene elements nee expression Vectors: Types  Plasmids As Vectors Plasmids are circular DNA molecules found in bacteria as extrachromosomal elements are replicated by the host’s machinery independently of t Natural plasmids have been modified to be used as vecto All plasmids are derived from the original plasmid pBR3 Most plasmids are between 2-8 kilobase pairs (kb) in size Accommodate up to 10 kb DNA insert  Artificial plasmids A basic artificial plasmid needs:  Origin of replication  Antibiotic resistance or selection markers  MCS or multiple cloning site For gene expression:  Active promoter => the plasmid is now designed as expression plasmid  Artificial plasmids A basic artificial plasmid needs:  Origin of replication Ensures plasmid maintenance and repartition for daughter cells Controls plasmid copy number  Artificial plasmids Antibiotic resistance or selection markers  Artificial plasmids MCS or multiple cloning site Is a polylinker region introduced artificially into the plasmid Contains multiple restriction enzymes sites that allows vector digestion Site of insertion of the foreign DNA fragments  Artificial plasmids Active promoter If the gene is required to be expressed in the cells into proteins  Introduction of promoter and terminator upstream and downstream  Promoters and terminators commonly isolated from viruses or phages such as SP6, T7 polymerase or T3 polymerase  Artificial plasmids Other screening tools  Insertional inactivation of ß- galactosidase enzyme  Gene is inserted in a way to disrupt ß-galactosidase gene (LacZ)  ß-galactosidase gene cleaves chromogenic substrate X-gal into a blue product  Colonies without insert = blue  Colonies with insert = white  Artificial plasmids Other screening tools  Insertional inactivation of ß- galactosidase enzyme  Gene is inserted in a way to disrupt ß-galactosidase gene (LacZ)  ß-galactosidase gene cleaves chromogenic substrate X-gal into a blue product  Colonies without insert = blue  Colonies with insert = white  Artificial plasmids Other screening tools Insertional inactivation of ß-galactosidase enzyme PLASMIDS Can Be Specialized For Differen Reporter genes can be used to measure promoter activity or tis expression. Courtesy of Joachim Goedhart, Molecular Cytolog of Amsterdam. Stowers Institute for Medical Research Photo courtesy of Robb Krumlauf, Figure 03.10A: (a) Since the di GFP, derivatives that fluore Figure 03.09: Expression of a lacZ gene can different colors have been en be followed in the mouse by staining for b- galactosidase (in blue).  The Major Limitation of Cloning in Plas  Upper limit for clone DNA size is 12 kb  Cannot enter the cells without special transformation met  Inefficient for generating genomic libraries as overlappin to place in proper sequence  Preference for smaller clones to be transformed  If it is an expression vector there are often limitations reg protein expression  Other Vectors BACs (Bacterial artificial chromosomes) Large low copy number plasmids (have ori and sele marker) Can be electroporated into E. coli Useful for sequencing genomes, because insert size YAC (Yeast Artificial Chromosome) Can be grown in E.coli and Yeast Miniature chromosome (contains ori, selectable ma telomeres, and a centromere Can accept 200 kb -1000 kb; useful for sequencing  Cloning techniques There are three prerequisites for cloning genes in a new host.  First, there needs to be a method for introducing the DNA of i potential recipient. The methods available include transformati and electroporation The introduced DNA needs to be maintained in the new host. E function as a replicon in its new environment or it has to be int chromosome or a pre-existing plasmid. Finally, the uptake and maintenance of the cloned genes will o they are expressed.  Cloning techniques  The strains of E. coli used in gene manipulation are not naturally  Rather, competence is induced by chemically treating the cells  Transformation => DNA uptake by the cells  Competence => ability of cells to uptake DNA  Bacterial transformation methods  Chemical transformation  Electroporation  Transduction (using phages)  Bacterial Transforma Traditional method involves incubat bacterial cells in concentrated cal salt solution => The solution makes cell membrane leaky, permeable to t plasmid DNA Transformation Procedu http://www.phschool.com/science/biology_place/labbench/lab6/concepts1.html Results Electroporation  Important Terms Transformation- Transfer of genetic m bacterial and plant cells Transfection-Transfer of non-viral ge material into eukaryotic cells. Infection/ Transduction- Transfer of genetic material into cells.  Important Terms Stable transfectants Cells that have integrated foreign their genome. Suicide vectors Integrative vectors Transient transfectants  Foreign DNA does not integrate in genome but genes are expressed for time (24–96 hours). Plasmids with Ori  Gene transfer into animal cell Introduction of DNA into somatic cells Somatic cells are limited in development Can’t be used to create transgenic animals Used to produce recombinant proteins : vacci hormones…. Or for basic research Introduction of DNA into germinal cell embryos, isolated germ cells or gametes) Note that transgenic animals can be achie manipulation Embryonic Stem cells or som by special techniques Gene transfer into somatic Direct transfer by physical transfection Chemical mediated transfection in which the persuaded to take up DNA by endocytosis Introduction of DNA packaged inside a virus (Transduction) Introduction of DNA packaged inside a bacter (bactofection) Applications of recombina DNA Production of eukaryotic proteins bacteria Gene therapy Example of SCID gene therapy Mutagenesis Gene targeting using CRISPR-CAS sys RNA interference Production of eukaryotic pr in bacteria Valuable proteins that could be isolat eukaryotes only in small amounts and a expense can now be produced in large q in genetically engineered bacteria. Proteins such as human insulin and hum hormone are valuable pharmaceuticals u treat diabetes and pituitary dwarfism, respectively. Expression of Human Growth Ho in E. coli Gene therapy  Gene therapy = Introduction of normal gen that contain defective genes to reconstitute protein product  GT is used to correct a deficient phenotype sufficient amounts of a normal gene produ synthesized  to improve a genetic disord  Transgenic Human = gene therapy  Modification of somatic cells by transferring sequences into the genome.  Somatic cells necessary to ensure that inse not carried over to the next generation.  DIFFERENTS APPROACHES EX VIVO * Cells modificaions in an appropriated lab – certificate *potential g irradiation IN VIVO * Direct administration of transfer gene vecto * problems in targeting cells to be modified  Gene therapy Gene transfer Viral vectors Retroviruses Adenoviruses Non viral vectors Liposomes DNA-protein conjugates  The First Case The first gene therapy was performed on Septe 1990 Ashanti DeSilva was treated for SCID Sever combined immunodeficiency Doctors removed her white blood cells, inserted the m into the WBC, and then put them back into her blood This strengthened her immune system Only worked for a few months  Current Status of gene therapy Only three FDA approved human gene therapy product for s Reasons: In 1999, 18-year-old Jesse Gelsinger died from multiple org days after treatment for omithine transcarboxylase deficie Death was triggered by severe immune response to adenovirus January 2003, halt to using retrovirus vectors in blood stem because children developed leukemia-like condition after suc treatment for X-linked severe combined immunodeficiency d Requirements for Approval o Gene-Therapy Protocol The gene must be cloned and well charac An effective method must be available f delivering the gene into the desired tis or cells. The risks of gene therapy to the patien have been carefully evaluated and shown minimal. The disease must not be treatable by ot strategies. Data must be available from preliminary experiments with animal models or human and must indicate that the proposed gene therapy should be effective.  ADA-SCID is a Good Candi Disease for Gene Thera The disease gene is cloned and charact White blood cells can easily be obtain ADA-SCID patients and reintroduced. A small amount of functional ADA will partial immune function. Overproduction of ADA does not have to effects. The ADA Retroviral Gene Transfer Vector Recom retro produ labor - Hav gen - Don gen res pat  Mutagenesis Reverse genetics change specifically a known DNA sequence advances in sequencing) and detect the r phenotype Remove the gene completely => Knock- Replace the gene with another mutant selectable marker => knock-in Reduce the expression of the gene at level => Knock-down by RNA interferen Gene knockout in the mouse Gene knockout in the mouse Gene knockout in th Recombinant DNA technology mouse Modification by gene editing: CRISPR/CAS What Is CRISPR system? CRISPR derives its name from "clustered reg interspaced short palindromic repeats” = ge sequences that microbes use to defend thems against viral attacks. They are associated with proteins called Ca associated bacteria use the sequences to recognize and future invading viruses Only in prokaryotes Scientists have adopted this system for use CRISPR proteins act like scissors, cutting DNA. When DNA is cut, the c Scientists can give cells healthy pieces of DNA to use for repair templa DNA so it has a healthy sequence.  Component CAS9 Guide DN spacer DN linked to t complex s Host DNA the (genom fragment o  Without the repair DNA bases durin  In the prese the target D DNA will be cleavage.  Rna interference  A series of recent discoveries has revealed t class of RNAs called noncoding RNAs are even than previously imagined.  Such RNAs play widespread roles in regulating expression and in protecting the genome from transposable elements.  Two types:  Long non coding RNAs (lncRNAs)  Small non coding RNAs  microRNAs (miRNAs)  Small interfering RNAs (siRNAs)  Piwi-interacting RNAs (piRNAs) Rna interference  Rna interference  short single-stranded RNAs (20–30 nucleotides) guide RNAs that selectively reorganize and bin base-pairing—other RNAs in the cell: a process RNAi or RNA interference  When the target is a mature mRNA, the small no can:  inhibit its translation  catalyze its destruction.  If the target RNA molecules in the proces transcribed, the small noncoding RNA can bi direct the formation of certain types of re chromatin on its attached DNA template Rna interference  Knock down = Rnai The addition of double-stranded RN animal and plant cells reduces the expression of the gene from which t double-stranded RNA sequence is der This “gene silencing,” which speci reduces the concentration of a targ by up to 90%, is reversible, since no change in the target cells’ DNA.  This phenomenon has been termed R interference (or “RNAi”) and occurs naturally in virtually all eukaryot organisms and has been used to knoc specific genes Knock Rn  Gene mapping an Positional cloni Why positional cloning? The ability of scientists to identify a isolate genes based on information abou location in the genome was one of the f major contributions of genomics researc In principle, this approach, called pos cloning, can be used to identify and cl gene with a known phenotypic effect in species. Positional cloning has been used extens many species, including humans to detec responsible for diseases  Why mapping and cloning g Locate causes of genetic disorders Determine identity of gene product We wil the us positi Determine how mutations cause theirto ide effect human respon Develop new diagnostic techniques Huntin diseas fibros Develop new therapies Human genome- Sequencing, mapping, Huntignton’s disea Huntingtin disease Huntington’s disease (HD) is genetic d caused by an autosomal dominant mutati occurs in about one of every 10,000 in of European descent. Individuals with HD undergo progressiv degeneration of the central nervous sy usually beginning at age 30 to 50 year terminating in death 10 to 15 years la To date, HD is untreatable. Huntingtin disease identification of the gene and the mut defect responsible for HD has kindled an effective treatment in the future. Because of the late age of onset of th disease, most HD patients already have before the disease symptoms appear. Since the disorder is caused by a domi mutation, each child of a heterozygous patient has a 50 percent chance of bei afflicted with the disease. These children observe the degeneratio death of their HD parent, knowing that have a 50:50 chance of suffering the s  Huntingtin gene mapping The gene responsible for HD (HTT, for h was one of the first human genes shown tightly linked to a restriction fragmen polymorphism (RFLP). In 1983, James Gusella, Nancy Wexler, a demonstrated that the HTT gene cosegreg RFLP that mapped near the end of the sh chromosome 4. They based their findings mainly on dat studies of two large families, one in V one in the United States The task of finding the causative gene 10 years to accomplish.  Huntingtin gene Gusella, Wexler, and coworkers iden gene, first called IT15 (for Intere Transcript number 15) and subsequen huntingtin, that spans about 210 kb end of the short arm of chromosome This gene contains a trinucleotide (CAG)n, which is present in 11 to 3 on each chromosome 4 of healthy ind In individuals with HD, the chromos carrying the HTT mutation contains or more copies of the CAG repeat in gene.  Huntingtin gene Moreover, the age of onset of HD is negatively with the number of copies of the trinucleotide Rare juvenile onset of the disease occurs in c unusually high repeat copy number. The trinucleotide repeat regions of HTT genes with repeat numbers often expanding and someti between generations. Gusella, Wexler, and collaborators detected ex repeat regions in chromosomes from 72 differen HD, leaving little doubt that they had identif gene. How to diagnose today the Expanded Trinucleotide Repe huntingtin ? How to diagnose today the Expanded Trinucleotide Repe huntingtin ? Cystic fibrosis disease Cystic Fibrosis Autosomal recessive Phenotypes Salty sweat Accumulation of mucus in lungs, pancreas, Frequent respiratory infections The CF gene was identified by position cloning.  Information Used to Map t Gene RFLP mapping localized the gene to a 50 of chromosome 7. Chromosome walks and jumps allowed cons a detailed map of the region. Three CpG islands are located upstream gene. Zoo blots demonstrated conservation of other species. A cDNA library prepared from sweat glan screened with exon sequences. Mapping the CF Gene  Chromosome walking Positional cloning is accomplished by mapping interest, identifying an RFLP, VNTR, STR, or o marker near the gene, and then “walking” or “j the chromosome until the gene is reached. Chromosome walks are initiated by the selectio marker (RFLP or known gene clone) close to the interest and the use of this clone as a hybrid screen a genomic library for overlapping seque Chromosome walking Restriction maps are constructed for the ove clones identified in the library screen, and restriction fragment farthest from the origi is used to screen a second genomic library c by using a different restriction enzyme or t a library prepared from a partial digest of DNA. Repeating this procedure several times and i series of overlapping genomic clones allow a to walk along the chromosome to the gene of The CF Gene Encodes the Channel CF is caused by loss of functi conductance regulator, the CF The gene codes for a chloride In a European survey of over most frequent mutations were N1303K, R117H, W1282X, an Splice site) These variants accounted for F508del present in 66.7% of t Mutations in the CF Gene The first and mutation in C removes a ph position 508 o More than 1,9 variations hav around the CF populations. A list of mutat references is Genome Vari http://www.ge Molecular diagnosis of CF At a minimum panel should panel of 23 recommende College of M Sequencing p and more rar differences a expressivity i mutations to b