Genomics and Proteomics, Study notes of Genomics

Download Genomics and Proteomics and more Study notes Genomics in PDF only on Docsity! GENOMICS AND PROTEOMICS Every organism possesses a genome that contains the biological information needed to construct and maintain a living example of that organism. Most genomes, including the human genome and those of all other cellular life forms, are made of DNA (deoxyribonucleic acid), but a few viruses have RNA (ribonucleic acid) genomes. DNA and RNA are polymeric molecules made up of chains of monomeric subunits called nucleotides. Each molecule of DNA comprises two polynucleotides wound around one another to form the famous double helix, in which the two strands are held together by chemical bonds that link adjacent nucleotides into structures called base pairs. The human genome, which is typical of the genomes of all multicellular animals, consists of two distinct parts • The nuclear genome comprises approximately 3,235,000,000 base pairs of DNAs, divided into 24 linear molecules, the shortest 48,000,000 base pairs in length and the longest 250,000,000 base pairs, each contained in a different chromosome. These 24 chromosomes consist of 22 autosomes and the two sex chromosomes, X and Y. Altogether, some 45,500 genes are present in the human nuclear genome. • The mitochondrial genome is a circular DNA molecule of 16,569 base pairs, up to 10 copies of which are present in each of the energy-generating organelles called mitochondria. The human mitochondrial genome contains just 37 genes. Introduction: The genome is a store of biological information, but on its own it is unable to release that information to the cell. Utilization of the biological information contained in the genome requires the coordinated activity of enzymes and other proteins, which participate in a complex series of biochemical reactions referred to as genome expression. The initial product of genome expression is the transcriptome, a collection of RNA molecules derived from those genes that are active in the cell at a particular time. The transcriptome is maintained by the process called transcription, in which individual genes are copied into RNA molecules. The second product of genome expression is the proteome, the cell’s repertoire of proteins, which specifies the nature of the biochemical reactions that the cell can carry out. The proteins that make up the proteome are synthesized by translation of some of the individual RNA molecules present in the transcriptome. Genomics: The term genomics was first used by Thomas Roderick in 1986. It refers to the study of structure and function of entire genome of a living organism. Genome refers to the basic set of chromosomes. In a genome, each type of chromosome is represented only once. Now genomics is being developed as a sub discipline of genetics which is devoted to the mapping, sequencing and functional analysis of genomes. • It is a computer aided study of structure and function of entire genome of an organism. • It deals with mapping and sequencing of genes on the chromosomes. • It is a rapid and accurate method of gene mapping. It is more accurate than recombination mapping and deletion mapping techniques. • The genomic techniques are highly powerful, efficient and effective in solving complex genetic problems. • Now use of genomic techniques has become indispensible in plant breeding and genetics. Types of Genomics: The discipline of genomics consists of two parts, viz. structural genomics and functional genomics. These are defined as : Structural Genomics: It deals with the study of the structure of entire genome of a living organism. In other words, it deals with the study of the genetic structure of each chromosome of the genome. It determines the size of the genome of a species in mega-bases [Mb] and also the genes present in the entire genome of a species. Functional Genomics: The study of function of all genes present in the entire genome is known as functional genomics. It deals with transcriptome and proteome. The transcriptome refers to complete set of RNAs transcribed from a genome and proteome refers to complete set of proteins encoded by a genome. Preparation of four Reaction Mixture G- Tube To the first test tube DMS ( Dimethyl sulphate ) is added which methylate Purines that is G& A. Methylation of G is 5 times more then the A. This procedure works only one purine per strand methylated. After that Piperidine is added to the sample and it will cleave at the point G. A & G Tube To the and test tube DMS along with formic Acid are added to the sample. Then Piperidine is added to cleave the DNA at the point A &G. C & T- Tube This sample is treated with Hydrazine and a dilute buffer along with Piperidine. Piperidine cleaves at the point C&T. C- Tube This sample is treated with hydrazine with 2 Molar NaCI. As a result, by the action of Piperidine .It can cleave at the point C. The reactions would be loaded on high percentage polyacrylamide gels and the fragments resolved by electrophoresis. The gel would then be transferred to a light-proof X-ray film cassette, a piece of X-ray film placed over the gel, and the cassette placed in a freezer for several days. Wherever a labeled fragment stopped on the gel the radioactive tag would expose the film due to particle decay (autoradiography). Since electrophoresis, whether in an acrylamide or an agarose matrix, will resolve nucleic acid fragments in the inverse order of length, that is, smaller fragments will run faster in the gel matrix than larger fragments, the dark autoradiographic bands on the film will represent the 5’→3’ DNA sequence when read from bottom to top. Disadvantage: • It requires extensive use of hazardous chemicals. • It has a relatively complex set up / technical complexity. • It is difficult to “scale up” and cannot be used to analyze more than 500 base pairs. Chain Termination Method (Sanger Dideoxy Method): This method was developed by Fred Sanger in 1974, which utilise DNA polymerase to extend DNA chain length. Sanger used the principle of DNA replication for the development of Dideoxy Sequencing method. At about the same time as Maxam-Gilbert DNA sequencing was being developed; Fred Sanger was developing an alternative method. Rather than using chemical cleavage reactions, Sanger opted for a method involving a third form of the ribose sugars. Ribose has a hydroxyl group on both the 2’ and the 3’ carbons whereas deoxyribose has only the one hydroxyl group on the 3’ carbon which is used for DNA synthesis. There is a third form of ribose in which the hydroxyl group is missing from both the 2’ and the 3’ carbons. This is dideoxyribose. Sanger knew that whenever a dideoxynucleotide was incorporated into a polynucleotide, the chain would irreversibly stop, or terminate and (dents) is used as DNA chain terminators. The enzymatic synthesis method requires 5 things 1. A short chain primer 2. A ssDNA templates 3. The 4 terminators (ddATP, ddGTP, ddTTP, ddCTP) 4. The four NTPs 5. A DNA Polymerase Primer: A primer is a short nucleic acid sequence which acts as a starting point for the DNA polymerases enzymes for the complementary strand synthesis. Primer is generally radiolabelled to generate the autoradiograph. The label may also introduce into the new strand by adding radiolabelled deoxynucleotides (P32). Template DNA: The ssDNA to be sequenced is called template DNA. The template DNA is used by DNA polymerase to attach complementary bases during new DNA synthesis. Polymerase: DNA polymerase is a type of enzyme that responsible for forming new copies of DNA. In this method DNA polymerase 1 is used. Process & Technique 1. The DNA fragments to be sequenced is denatured and a single strand of DNA is used as template DNA for replication. 2. This reaction needs a primer for initiation & usually this is a chemically synthesized Oligonucleotides. 3. A reaction mixture is prepared by adding Template DNA + Primer + DNA Polymerase 1 + deoxynucleotide ( dATP ,dGTP,dCTP,dTTP) . 4. The reaction mixture is divided into 4 tubes with having low concentration of dideoxynucleotide ( dd ATP , dd GTP, dd CTP , dd TTP) respectively. The dd NTPs act as terminator of chain elongation because the 3' end lacks a hydroxyl group. 5. After suitable incubation Period again heated for the denaturation of DNA & that samples are electrophoresed in polyacrylamide gel. 6. The radioactive bands of ss DNA are detected by autoradiography. Reaction-2: (ATP as a cofactor for enzyme Luciferase) The second major molecule is Luciferin. Luciferin oxidized to oxyluciferin in the presence of ATP produced from reaction 1. In the oxidised state luciferin produced some amount of light and that light is measured to detect the presence of pyrophosphate group. Working Principle: In the procedure of pyrosequencing each dNTPS is added individually along with nucleotidase enzyme. This enzyme degrades the dNTPs if it is not incorporated in to the newly synthesized strand. Once the appropriate nucleotide is incorporated in to the new DNA strand, a molecule of Pyrophosphate is released. This can convert to ATP by sulfurase enzyme then into a light by luciferase enzyme. The amount of the light released by the enzymatic reaction is detected by the charged device (detector). Note: Addition of dATPαS (Deoxyadenosine thiotriphosphate) is used instead of dATP. Because free dATP attach with luciferase and give false signal. The dATPαS is recognised by DNA pol. Enzyme only. Not by Luciferase. E amp - 1 v 3 ——ATGGCCCTATTA~S Te ‘ DNA Poimet pol. Enzyme Nucleotiad a8 Aigbh- Nucleotides | Xyft- A ATPAS — A ATPAS +t ATTP + a TTP + d ctTP = al CTP — ad GP = AGTP = ad ATP aS + aA ATPKS | ft atte = dTTP + ad CTP ++ |dctp 7 al GTP +44 [dar = x 44 3 G& Ss ws ec AA a T A AT T a3 A 1CG ATCG ATCG ATCC sfaclectide —  SHOTGUN & HIERARCHICAL (CLONE CONTIG) Sanger DNA sequencing, only works for a certain distance beyond the sequencing primer (best from about 30 nt to 350 nt; the “read length”). Beyond that, very few products are produced because chain termination has already occurred. Therefore, to sequence a longer DNA, special methods are required. Another approach, used to sequence very large amounts of DNA (such as an entire genome), is shotgun sequencing. In this strategy, the DNA is first shredded into smaller fragments which can be sequenced individually. The sequences of these fragments are then reassembled into their original order, based on overlaps, ultimately yielding the complete sequence. “Shredding” of the DNA can be done using restriction enzymes, or mechanically by shearing. Alignment of the sequences of overlapping pieces is done by computer. In the case of the human genome project, a massive amount of data is involved, requiring supercomputer technology. Methods of Genome Sequencing 1. Hierarchical Shotgun Method: Useful for sequencing genomes of higher vertebrates that contain repetitive sequences. 2. Whole genome Shotgun Sequencing: -Useful for smaller genomes Hierarchical Shotgun Method: This method was preferred by the human genome project. That is also know as ◼ Clone contigs Method ◼ Clone by Clone methods ◼ Map Based methods Working principle: • The whole is mapped with some specific stain and identify the gene carrying region. • The genome is split into larger fragments (50-200kb) using restriction/cutting enzymes. • These fragments are cloned in bacteria (E. coli) using BACs (Bacterial Artificial Chromosomes), where they are replicated and stored. • The BAC inserts are isolated and the whole genome is mapped by finding markers. • Each BAC fragment is fragmented randomly into smaller pieces (reads) and these fragments are individually sequenced using automated Sanger sequencing and pyrosequencing methods. • These sequences are aligned so that identical sequences are overlapping. • Assembly of the genome is done on the basis of prior knowledge of the marker. • A computer based programme stiches the sequence using algorithims.