









Study with the several resources on Docsity
Earn points by helping other students or get them with a premium plan
Prepare for your exams
Study with the several resources on Docsity
Earn points to download
Earn points by helping other students or get them with a premium plan
Community
Ask the community for help and clear up your study doubts
Discover the best universities in your country according to Docsity users
Free resources
Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors
The notes have been prepared by a thorough reading of the book and understanding of lectures.
Typology: Study notes
1 / 15
This page cannot be seen from the preview
Don't miss anything!
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
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.
2. Denaturation of DNA (ds DNA → ss DNA) DimethylSulfoxide ( DMSO ) is added to DNA sample & heated to 90°c. This breaks the base pairing and enabling them to be separated from one another by gel electrophoresis. One strand is purified from the gel and divided into four samples. 3. Chemical Degradation of SS DNA. The sample is divided into 4 test tubes & each treated with different reagent having the property of destroying either only G or only C Or A & G / T& C. This chemical degradation method is a two-step catalytic process involving piperidine and two chemicals that selectively attack purines and pyrimidines. Purines will react with dimethyl sulfate and pyrimidines will react with hydrazine in such a way as to break the glycoside bond between the ribose sugar and the base. Piperidine will then catalyze phosphodiester bond cleavage where the base has been displaced. Moreover, dimethyl sulfate and piperidine alone will selectively cleave guanine nucleotides but dimethyl sulfate and piperidine in formic acid will cleave both guanine and adenine nucleotides. Similarly, hydrazine and piperidine will cleave both thymine and cytosine nucleotides whereas hydrazine and piperidine in 2 M NaCl will only cleave cytosine nucleotides.
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 enzymatic synthesis method requires 5 things
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.
Genome sequence assembly refers to aligning & merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed because DNA Sequencing technology can't read whole genomes in one go,but rather reads small pieces of DNA between 20 - 2000 base paris depending up on the technology used. The early attempts at reconstructing genome sequence relied on molecular techniques to sequence overlapping genomic segments. The order and position of the corresponding sequence of reads were defined by the experimental methods & gluing together into a complete genome sequence was easily done without computer but it was time consuming. It quickly became clear that reconstructing genome sequencing would require computer programme. The first sequence assemblers —software packages that convert sequence reads into contigs—simply compared pairs of reads, merged those with the longest overlaps, and repeated the process until no further overlaps were present. The result is an overlap graph , from which a master sequence can be assembled. This is the Greedy Graph-based Assemblers. The de Bruijn Graph Approach More recently developed sequence assemblers take an alternative approach that is less computationally intensive and hence is applicable to next-generation sequence reads. These assemblers make use of a representation called a De Bruijn graph, which is a mathematical concept for identifying overlaps between strings of symbols. The strings must be of equal length, so when this method is used in sequence assembly, the initial step is to break the sequence reads into smaller segments or k - mers, typically 20– 30 nucleotides in length. Duplicate k - mers are discarded, so this step reduces the size of the data set. Each k - mer is then converted into a prefix sequence (the k - mer minus its last nucleotide) and a suffix sequence (the k - mer minus its first nucleotide). Overlaps between k-mers are then identified by searching for pairs where the suffix of one k - mer is identical to the prefix of the second k - mer. Once all overlaps are identified, the k - mers are linked together as a De Bruijn graph.The master sequence can then be read from the graph.