Gene Expression Technologies AZAR-1403 PDF

Gene expression technologies 1 AZAR-1403  Cloning and Expression 2  Cloning and gene expression technologies are used by researchers in diverse fields including :  gene function  molecular pathway analys...

Gene expression technologies 1 AZAR-1403  Cloning and Expression 2  Cloning and gene expression technologies are used by researchers in diverse fields including :  gene function  molecular pathway analysis  embryonic development  disease research  biotechnology research for the production of antibodies  small molecule identification.  Gene expression 3  Gene expression is the process by which a gene gets turned on in a cell to transcript RNA and produce proteins.  The first step in expression is transcription of the gene into a complementary RNA strand.  RNA, or ribonucleic acid, plays a crucial role in various biological processes, particularly in protein synthesis.  Some genes, such as those coding for transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, the transcript itself is the functionally important molecule.  For other genes, the transcript is translated into a protein molecule.  Control of Gene Expression 4  Controlling gene expression is often occur by controlling transcription initiation.  The product of a regulatory genes is required to initiate (turn on) or (turn off) the expression.  Regulatory genes → regulatory proteins that bind to DNA to either block or stimulate transcription, depending on how they interact with RNA polymerase. 5  Types of gene expression 6 a. Constitutive expression: Some genes are essential and necessary for life, therefore are continuously expressed. These genes are called housekeeping genes (such as: Actin, GAPDH(glyceraldehyde -phosphate dehydrogenase). b. Induction and repression: The expression levels of some genes fluctuate in response to the external signals.  Gene Expression 7  Every cell in an organism's body contains the same set of genes, only a fraction of these genes are used in any given cell at any given time.  It is carefully controlled pattern of what is called "gene expression".  It makes a liver cell different from a muscle cell, and a healthy cell different from a cancer cell.  But how can we determine which genes are "turned on" and when?  Gene Control in Prokaryotes 8  One way in which prokaryotes control gene expression is one group functionally related genes together so that they can be regulated together. This grouping is called an operon. An operon consists of: 1- a promoter (binding site for RNA polymerase) 2- an operator (repressor binding site that overlaps the promoter). 3- structural genes.  Regulatory region 9 Promoter: is a specific short sequence on DNA at which RNA polymerase attaches and initiates transcription at the beginning of the transcription unit. Operator: is a specific short of DNA sequence adjacent to the structural genes that the repressor protein* can bind to and prevent the transcription of structural genes. *Repressor proteins encoded by repressor genes to regulate gene expression. Activators: the activity of RNA polymerase is also regulated by interaction with accessory proteins called activators.  The presence of the activator removes repression and transcription occurs.  Eukaryotic cells modify RNA 10  Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm.  At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap. 1. protect mRNA from hydrolytic enzymes. 2. a translation start point for ribosomes.  At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail. 1. Protect mRNA from degradation by exonucleases. 2. Facilitate export of mRNA from the nucleus. 3. Terminate transcription. In eukaryotes, proteins called transcription factors recognize the promoter region, especially a TATA box, and bind to the promoter. 11 After they have bound to the promoter, RNA polymerase binds to transcription factors to create a transcription initiation complex. RNA polymerase then starts transcription.  How do we measure gene expression? 12  Gene expression is dynamic, and the same gene may act in different ways under different circumstances.  Why two organisms have similar genotypes but different phenotypes.  What is the cause of this variation in phenotype?  Could the difference stem from differing regulation of gene expression?  To go about answering these types of questions, we need to use laboratory techniques.  Expressing Cloned Eukaryotic Genes 13  After a gene has been cloned, its protein product can be produced in larger amounts for research.  Cloned genes can be expressed as protein in either bacterial or eukaryotic cells.  Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells  Because of differences in promoters and other DNA control sequences.  Molecular biologists using eukaryotic cells, such as yeasts, cultured mammalian or insect cells as hosts for cloning and expressing genes.  Scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter  What is an expression vector 14  It is a plasmid or virus that is specially designed for expressing genes in a cell.  They have basic features of a vector like:  Ori (origin of replication)  Unique insertion site  Selectable marker  regulatory elements  Translational initiation regions  Translational terminator  Analyzing Gene Expression 15  The simplest procedures for studying transcribed sequences are based on hybridization analysis.  Nucleic acid probes can hybridize with mRNAs transcribed from a gene , Probes can be used to identify where or when a gene is transcribed in an organism.  Northern hybridization  Zoo-blotting  hybridization between gene and RNA Northern hybridization Northern blotting was developed by James Alwine and George Stark at Stanford University (1979). 16  Northern blotting Workflow 17 1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) 2. Sample’s are loaded on gel and the RNA samples are separated according to their size on an agarose gel.  Northern blotting Workflow 18 3. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper/ diazobenzyloxymethyl papers by creating the sandwich arrangement.  Northern blotting Workflow 19 4. The membrane is placed in a dish containing hybridization buffer with a labeled probe. 5. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 6. The membrane is washed to remove unbound probe.  Northern blotting Workflow 20 6. The labeled probe is detected via autoradiography. 7. Now the expression patterns of the sequence of interest in the different samples can be compared.  Zoo-blotting(garden blotting ) 21  Zoo blotting is a specialized technique used in molecular biology to compare the DNA sequences of different animal species, particularly focusing on protein-coding genes.  A hybridization analysis with poorly expressed in tissue-specific genes by searching related sequences in the DNAs of other organisms.  This approach, like homology searching.  Gene Conservation Studies.  Identifying Functional Genes.  Comparative Genomics.  hybridization between gene and RNA 22  The approximate length of a transcript can be measured by northern hybridization.  This method does not allow the start and end positions of the RNA molecule to be mapped onto the DNA sequence of the cloned gene.  To obtain this information , hybridization product formed between a cloned gene and its RNA.  Heteroduplex 23  Heteroduplex is formed between a DNA strand, containing a gene, and its mRNA.  The boundaries between the double and single stranded regions will mark the start and end points of the mRNA.  Introns, which are present in the DNA but not in the mRNA, will ‘loop out’ as additional single stranded regions.  S1 nuclease will digest the non hybridized single stranded DNA regions at each end of the DNA– RNA hybrid, along with any looped out introns.  The sizes of the protected DNA fragments could be measured by gel electrophoresis.  Primer extension 24  primer extension identify the 5′ end of an RNA.  Primer extension can only be used if at least part of the sequence of the transcript is known.  A short oligonucleotide primer must be annealed to the RNA at a known position.  Once annealed, the primer is extended by reverse transcriptase.  The resulting cDNA synthesis reaction continues until the end of the RNA transcript is reached.  Locating the position of this terminus on the DNA sequence is achieved simply by determining the length of the single stranded DNA molecule and correlating this information with the annealing position of the primer.  Complementary DNA(CDNA) 25  Northern hybridization and zoo-blotting enable the presence or absence of genes in a DNA fragment to be determined.  But give no positional information relating to the location of those genes in the DNA sequence.  The easiest way to obtain this information is to sequence the relevant cDNAs.  A cDNA is a copy of an mRNA and so corresponds to the coding region of a gene, plus any leader or trailer sequences that are also transcribed.  Comparing a cDNA sequence with a genomic DNA sequence therefore delineates the position of the relevant gene and reveals the exon-intron boundaries.  Preparing cDNA(RT-PCR) 26  It is quicker and more sensitive because it requires less mRNA than Northern blotting  Most eukaryotic mRNAs have a poly(A) tail at their 3′ end.  The primer is a short synthetic DNA oligonucleotide, typically 20 nucleotides in length, made up entirely of Ts (‘oligo(dT)’).  When the first strand synthesis has been completed, the preparation is treated with ribonuclease H, which specifically degrades the RNA component of an RNA-DNA hybrid.  The enzyme does not degrade all of the RNA, instead leaving short segments that prime the second DNA strand synthesis reaction, this one catalyzed by DNA polymerase I.  Rapid amplification of cDNA ends (RACE) 27  RACE is designed to amplify cDNA sequences from mRNA templates, focusing on the 3' or 5' ends of the transcripts.  This technique helps in identifying the complete sequence of mRNA, including regions that may not be known initially.  There are two primary types of RACE:  3' RACE: Targets the 3' end of mRNA, utilizing the natural poly(A) tail as a priming site for reverse transcription and subsequent PCR amplification.  5' RACE: Focuses on the 5' end of mRNA, often requiring additional steps to add linkers or adapters to facilitate amplification. 28  Identifying protein binding sites on a DNA molecule 29  A control sequence is a region of DNA that can bind a regulatory protein.  It should therefore be possible to identify control sequences upstream of a gene by searching the relevant region for protein binding sites.  There are three different ways of doing this.  Gel retardation of DNA–protein complexes  Footprinting with DNase I  Modification interference assays  Gel retardation of DNA–protein complexes 30  Proteins are quite substantial structures and a protein attached to a DNA molecule results in a large increase in overall molecular mass.  In practice a DNA fragment carrying a bound protein is identified by gel electrophoresis, as it has a lower mobility than the uncomplexed DNA molecule.  The procedure is referred to as gel retardation or the electrophoretic mobility shift assay (EMSA).  The electrophoretic mobility shift assay (EMSA) is a rapid and sensitive method to detect protein-nucleic acid interactions. Electrophoretic mobility shift assay (EMSA). 31  The region of DNA upstream of the gene being studied is digested with a restriction endonuclease  Then mixed with the regulatory protein  The restriction fragment containing the control sequence Forms a complex with the regulatory protein.  The location of the control sequence is then determined by finding the position on the restriction map of the fragment that is retarded during gel electrophoresis.  Footprinting with DNase I 32  The procedure generally called footprinting enables a control region to be positioned within a restriction fragment that has been identified by gel retardation.  Footprinting works on the basis that the interaction with a regulatory protein protects the DNA in the region of a control sequence from the degradative action of an endonuclease such as DNase I.  This phenomenon can be used to locate the protein binding site on the DNA molecule.  Sequencing  Footprinting with DNase I 33  The DNA fragment being studied is first labelled at one end, and then complexed with the regulatory protein.  Then DNase I is added, but the amount used is limited so that complete degradation of the DNA fragment does not occur.  After removal of the bound protein and separation on a polyacrylamide gel, the family of labelled fragments appears as a ladder of bands.  Their absence shows up as a ‘footprint’.  The region of the DNA molecule containing the control sequence can now be worked out from the sizes of the fragments on either side of the footprint.  Modification interference assays(MIA) 34  Gel retardation analysisٍ)EMSA) and footprinting enable control sequences to be located, but do not give information about the interaction between the binding protein and the DNA molecule.  Nucleotides that actually form attachments with a bound protein can be identified by the modification interference assay.  Modification interference assays 35  As in footprinting, the DNA fragments must first be labelled at one end.  Then they are treated with a chemical that modifies specific nucleotides, an example being dimethyl sulphate (DMS), which attaches methyl groups to guanine nucleotides.  The DNA is mixed with the protein extract.  The binding protein will probably not attach to the DNA if one of the guanines within the control region is modified.  The products of piperidine treatment are now separated in a polyacrylamide gel and the labelled bands visualized.  The sizes of the bands that are seen indicate the position in the DNA fragment of guanines whose methylation prevented protein binding.  These guanines lie within the control sequence.  The modification assay can now be repeated with chemicals that target A, T, or C nucleotides to determine the precise position of the control sequence. Methylation interference assays 36  Identifying control sequences by deletion analysis 37  Gel retardation, footprinting, and modification interference assays are able to locate possible control sequences upstream of a gene, but they provide no information on the function of the individual sequences.  Deletion analysis is a totally different approach that not only can locate control sequences, but importantly also can indicate the function of each sequence.  The reporter gene assay 38  Cloning the gene that is being studied back into its host should not cause a problem.  The difficulty is that in most cases the host will already possess a copy of the gene within its chromosomes.  How can changes in the expression pattern of the cloned gene be distinguished from the normal pattern of expression displayed by the chromosomal copy of the gene?  The answer is to use a reporter gene.  The reporter gene assay 39  This is a test gene that is fused to the upstream region of the cloned gene ,replacing the latter.  When cloned into the host organism, the expression pattern of the reporter gene should exactly mimic that of the original gene, as the reporter gene is under the influence of exactly the same control sequences as the original gene. Studying the Expression of Interacting Groups of Genes 40  Automation has allowed scientists to measure the expression of thousands of genes at one time.  DNA microarray assays.  SAGE (serial analysis of gene expression).  RNA Sequencing  DNA microarray assays 41  DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions 42  DNA microarray assays 43  The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent markers are attached to these DNA fragments.  These are then allowed to react with probes of the DNA chip.  Then the target DNA fragments along with complementary sequences bind to the DNA probes  The remaining DNA fragments are washed away.  The target DNA pieces can be identified by their fluorescence emission by passing a laser beam.  A computer is used to record the pattern of fluorescence emission and DNA identification.  Serial analysis of gene expression(SAGE) 44  SAGE identifies and counts the mRNA transcripts in a cell with the help of short snippets of the genetic code, called tags.  These tags, enable researchers to match an mRNA transcript with the specific gene that produced it.  In most cases, each tag contains enough information to uniquely identify a transcript.  The name "serial analysis" refers to the fact that tags are read sequentially as a continuous string of information.  SAGE technique 45  A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided that the tag is obtained from a unique position within each transcript  Sequence tags can be linked together to from long serial molecules that can be cloned and sequenced  Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript. 46 SAGE FLOWCHART 47  RNA-Seq (RNA sequencing) 48  RNA-Seq, uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment.  It allows profiling the transcripts in a sample in an efficient and cost- effective way.  RNA-seq allows for an unbiased sampling of all transcripts in a sample, rather than being limited to a pre-determined set of transcripts.  Illumina Sequencing Platforms 49  Illumina  Sequencing by Synthesis (SbS)  /NovaSeq/HiSeq/NextSeq/MiSeq Short read length (50 to 300 bp)   Selection driven by cost, precision, speed, number of samples and number of reads required   Consult with the Sequencing Core  Illumina  NovaSeq      Illumina  NextSeq      Illumina  MiSeq  RNA Sequencing (RNA-seq) 50 Basic steps: Extract and isolate RNA, then convert it to cDNA Fragmentation and size selection Addition of any linkers, adapters, or barcodes Next-generation sequencing (NGS) of reads Analysis of read sequences: Includes read QC, alignment to reference genome, counting of reads over features (e.g.genes), and within- and between-group comparisons and contrasts RNA-seq include:  Bulk RNA Sequencing  Single-cell RNA Sequencing Bulk RNA-seq 51 single-cell RNA-Seq 52