gene expression traditional methods

Gene regulation, History and Evolution , Traditional Methods:
Northern blot
quantitative reverse transcription PCR (qRTPCR)
serial analysis of gene expression(SAGE) and
DNA microarrays.
DNA Chip

1. FUNCTIONAL GENOMICS & PROTEOMICS Submitted By: Dhaval Chaudhary M.V.Sc . (Animal Genetics & Brreding ) Methods to Study Gene Expression

2. What is Gene Expression? Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA.

3. In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, i.e. observable trait. The genetic information stored in DNA represents the genotype, whereas the phenotype results from the "interpretation" of that information. Such phenotypes are often expressed by the synthesis of proteins that control the organism's structure and development, or that act as enzymes catalyzing specific metabolic pathways. All steps in the gene expression process may be modulated (regulated), including the transcription, RNA splicing, translation and post-translational modification of a protein.

4. The process of gene expression involves two main stages: Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase, and the processing of the resulting mRNA molecule Translation: the use of mRNA to direct protein synthesis, and the subsequent post-translational processing of the protein molecule. Some genes are responsible for the production of other forms of RNA that play a role in translation, including transfer RNA (tRNA) and ribosomal RNA (rRNA).

5. A structural gene involves a number of different components: Exons: Exons code for amino acids and collectively determine the amino acid sequence of the protein product. It is these portions of the gene that are represented in final mature mRNA molecule. Introns: Introns are portions of the gene that do not code for amino acids, and are removed (spliced) from the mRNA molecule before translation.

6. Control regions: Start site - A start site for transcription. A promoter - A region a few hundred nucleotides 'upstream' of the gene (toward the 5' end). It is not transcribed into mRNA, but plays a role in controlling the transcription of the gene. Transcription factors bind to specific nucleotide sequences in the promoter region and assist in the binding of RNA polymerases. Enhancers - Some transcription factors (called activators) bind to regions called 'enhancers' that increase the rate of transcription. These sites may be thousands of nucleotides from the coding sequences or within an intron. Some enhancers are conditional and only work in the presence of other factors as well as transcription factors. Silencers - Some transcription factors (called repressors) bind to regions called 'silencers' that depress the rate of transcription.

7. Transcription Transcription is the process of RNA synthesis, controlled by the interaction of promoters and enhancers. Several different types of RNA are produced, including messenger RNA (mRNA) , which specifies the sequence of amino acids in the protein product, plus transfer RNA (tRNA) and ribosomal RNA (rRNA) , which play a role in the translation process.

8. Transcription involves four steps: Initiation . The DNA molecule unwinds and separates to form a small open complex . RNA polymerase binds to the promoter of the template strand (also known as the 'sense strand' or 'coding strand'). The synthesis of RNA proceeds in a 5' to 3' direction, so the template strand must be 3' to 5'. Elongation . RNA polymerase moves along the template strand, synthesising an mRNA molecule. In prokaryotes RNA polymerase is a holoenzyme consisting of a number of subunits, including a sigma factor (transcription factor) that recognises the promoter. In eukaryotes there are three RNA polymerases: I, II and III. The process includes a proofreading mechanism. Termination . In prokaryotes there are two ways in which transcription is terminated. In -dependent termination , a protein is responsible for disrupting the complex involving the template strand, RNA polymerase and RNA molecule. In independent termination , a loop forms at the end of the RNA molecule, causing it to detach itself. Termination in eukaryotes is more complicated, involving the addition of additional adenine nucleotides at the 3' of the RNA transcript (a process referred to as polyadenylation ). Processing . After transcription the RNA molecule is processed in a number of ways: introns are removed and the exons are spliced together to form a mature mRNA molecule consisting of a single protein-coding sequence. RNA synthesis involves the normal base pairing rules, but the base thymine is replaced with the base uracil .

9. Translation In translation the mature mRNA molecule is used as a template to assemble a series of amino acids to produce a polypeptide with a specific amino acid sequence. The complex in the cytoplasm at which this occurs is called a ribosome . Ribosomes are a mixture of ribosomal proteins and ribosomal RNA (rRNA), and consist of a large subunit and a small subunit.

10. Initiation . The small subunit of the ribosome binds at the 5' end of the mRNA molecule and moves in a 3' direction until it meets a start codon (AUG). It then forms a complex with the large unit of the ribosome complex and an initiation tRNA molecule. Elongation . Subsequent codons on the mRNA molecule determine which tRNA molecule linked to an amino acid binds to the mRNA. An enzyme peptidyl transferase links the amino acids together using peptide bonds. The process continues, producing a chain of amino acids as the ribosome moves along the mRNA molecule. Termination . Translation in terminated when the ribosomal complex reached one or more stop codons (UAA, UAG, UGA).

11. The ribosomal complex in eukaryotes is larger and more complicated than in prokaryotes. In addition, the processes of transcription and translation are divided in eukaryotes between the nucleus (transcription) and the cytoplasm (translation), which provides more opportunities for the regulation of gene expression.

12. Gene regulation Gene regulation is a label for the cellular processes that control the rate and manner of gene expression. A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced. Some genes are expressed continuously, as they produce proteins involved in basic metabolic functions; some genes are expressed as part of the process of cell differentiation; and some genes are expressed as a result of cell differentiation.

13. Mechanisms of gene regulation include: Regulating the rate of transcription. This is the most economical method of regulation. Regulating the processing of RNA molecules, including alternative splicing to produce more than one protein product from a single gene. Regulating the stability of mRNA molecules. Regulating the rate of translation.

14. History and Evolution

15. Traditional Methods: Northern blot quantitative reverse transcription PCR ( qRTPCR ) serial analysis of gene expression(SAGE) and DNA microarrays.

16. Northern blot Northern blot analysis is a low throughput method that uses electrophoresis to separate RNA by size. The separated RNA is transferred onto a nylon membrane and immobilised to the membrane through covalent linkage by UV light or heat. A labelled short oligonucleotide sequence or probe that is complementary to a sequence in the target transcript is introduced onto the membrane and its hybridisation to the target transcript is detected via the use of X-ray.

17. The Northern blot procedure is useful for determining RNA size and to detect alternative splice products. However, Northern blot uses RNA without conversion into complementary DNA (cDNA), therefore, the quality of quantification is compromised by even low levels of RNA degradation. Northern blot has also relatively low sensitivity, due to non-specific hybridisation , requires the use of radioactivity and requires greater amounts of RNA compared to qRTPCR .

18. quantitative Reverse Transcription PCR ( qRTPCR ) qRTPCR involves the reverse transcription of the target RNA into cDNA followed by PCR of the cDNA to amplify the signal for detection. It is a fluorescence-based real-time reaction method that allows for detection and relative quantitation of target RNAs. The qRTPCR method has improved to enable high throughput multiplexed reactions to quantify multiple genes in a single reaction. However, the throughput capability of the current technology of qRTPCR remains on the order of only hundreds of known transcripts in one assay, and is not adept for transcriptome-wide gene expression analysis.

19. Serial Analysis of Gene Expression(SAGE) It is gene expression profiling method that involves creating cDNA which is biotin-labelled at the 3′-end of the cDNA. Short sequence tags of 14 or 21 bp that can uniquely identify specific transcripts are extracted using restriction enzymes ( Neisseria lactamica III). Nla III cleaves the cDNA at the 5′-end of every CATG site. The cleaved sequence closest to the 3′-end of the cDNA is isolated using streptavidin beads. This isolated sequence is shortened again to contain only the CATG and the next ten nucleotides. This final sequence is the SAGE tag. These tags are ligated and cloned into a vector which are Sanger sequenced to identify the sequence tags.

20. This method allows direct measurement of transcript abundance and comparison between multiple samples. SAGE does not require a prior knowledge of the transcript sequence and has been used for discovery of novel transcripts and alternative splice isoforms. Nonetheless, SAGE is a costly technique with a laborious cloning procedure.

21. DNA microarrays/ biochip/DNA chip The DNA microarray technology has superseded single-gene approaches, allowing the measurement of RNA expression levels of thousands of known or putative transcripts simultaneously and was further developed to characterize the gene expression profile of a complete eukaryotic genome ( Saccharomyces cerevisiae ) . DNA microarray technology has enabled comprehensive characterization and/or comparison of expression signatures of various cell types and disease phenotypes. Gene profiling with microarrays have provided evidence of molecular heterogeneity in cancer.

22. Despite the success of gene expression profiling using DNA microarrays in its contribution to the field of biology, the technique remains limited by its requirement of a prior knowledge of the genes of interest. To overcome this limitation, tiling arrays have been developed. Prior to the advent of massively parallel sequencing technology, tiling arrays was the method of choice to identify novel transcribed regions.

23. Principle of DNA microarray The principle of DNA microarrays lies on the hybridization between the nucleic acid strands. The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. For this, samples are labeled using fluorescent dyes. Complementary nucleic acid sequences between the sample and the probe attached on the chip get paired via hydrogen bonds. The non-specific bonding sequences while remain unattached and washed out during the washing step of the process. Fluorescently labeled target sequences that bind to a probe sequence generate a signal. The signal depends on the hybridization conditions (ex: temperature), washing after hybridization etc while the total strength of the signal, depends upon the amount of target sample present.

24. Requirements of DNA Microarray Technique There are certain requirements for designing a DNA microarray system, viz: DNA Chip Target sample (Fluorescently labelled) Fluorescent dyes Probes Scanner

25. Applications of DNA Microarray In humans, they can be used to determine how particular diseases affect the pattern of gene expression (the expression profile) in various tissues, or the identity (from the expression profile) of the infecting organism. Thus, in clinical medicine alone, DNA microarrays have huge potential for diagnosis. Discovery of drugs Diagnostics and genetic engineering Proteomics Functional genomics Gene expression profiling Toxicological research ( Toxicogenomics )

26. Advantages of DNA Microarray Provides data for thousands of genes in real time. Single experiment generates many results easily. Fast and easy to obtain results. Promising for discovering cures to diseases and cancer. Different parts of DNA can be used to study gene expression.

27. Disadvantages of DNA Microarray Expensive to create. The production of too many results at a time requires long time for analysis, which is quite complex in nature. The DNA chips do not have very long shelf life.

28. Thank You..