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DNA Sequencing

Reading the Blueprint of Life. DNA Sequencing. Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001. Genomic DNA. All living organisms, from bacteria to human beings, contain DNA in the nuclei of each of their cells.

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DNA Sequencing

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  1. Reading the Blueprint of Life DNA Sequencing

  2. Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001

  3. Genomic DNA • All living organisms, from bacteria to human beings, contain DNA in the nuclei of each of their cells. • The blueprint of life exist in the DNA. • DNA consists of four building blocks or nucleotides (A, C, G and T) • Extraction of useful information from genome sequence of all organisms relies on computer-aided data handling –i.e. Bioinformatics.

  4. DNA Preparation • Whole chromosomes are too large to deal with, so the DNA is broken into manageably-sized overlapping segments. • The DNA segments are cloned using bacteria and formed a library of DNA segments. • It is then denatured (ie. melted), so that the two strands split apart.

  5. DNA Amplification • In order to sequence a piece of DNA, first need to amplify it. This is sometimes done by a process called polymerase chain reaction (PCR).Denatured DNA is added to a mixture for PCR: - Primer, - DNA polymerase - Nucleotides including special ones called dideoxynucleotides. • The dideoxynucleotides are fluorescently tagged.

  6. Green: A Blue: C Yellow: G Red: T Here is a gel with 28 DNA samples: Sequencing using gel electrophoresis CGACG………… Small molecules move faster.

  7. Sequence Assembly Using Mapping • The segments were cut to look for common sequence landmarks in overlapping fragments. • These landmarks were used to fingerprint the fragments so that it was possible to identify the position of the fragments in the chromosome– basic strategy of Genome Mapping. So Genome Mapping is the process of determining • location of a specific gene in the genome/chromosome & • relative distances between genes on the chromosome.

  8. Genetic Mapping: determine the relative position between two genes on a chromosome. Physical Mapping: determines the absolute position of a gene on a chromosome. Types of mapping

  9. Genetic (Genetic Linkage) Mapping • The genetic linkage map shows the arrangement of genes and genetic markers along the chromosomes as calculated by the frequency with which they are inherited together. • A genetic map determines a distance between markers (gene for inherited traits) • The closer the markers are physically, the more likely they will be inherited together. • Markers on genetic linkage map must vary between individuals.

  10. Commonly Used DNA Markers • RFLP (Restriction Fragment Length Polymorphism): RFLP is defined by the presence or absence of a specific site, called a restriction site, for a bacterial restriction enzyme. This enzyme breaks apart strands of DNA wherever they contain a certain nucleotide sequence. • VNTR (Variable Number of Tandem Repeat Polymorphism): It occurs in non-coding regions of DNA and is defined by the presence of a nucleotide sequence that is repeated several times.

  11. Commonly Used DNA Markers • Microsatellite polymorphism: It defined by a variable number of repetitions of a very small number of base pairs within a sequence. The number of repeats for a given microsatellite may differ between individuals, hence the term polymorphism (the existence of different forms within a population). • SNP (Single Nucleotide Polymorphism): are individual point mutations, or substitutions of a single nucleotide, that do not change the overall length of the DNA sequence in that region. SNPs occur throughout an individual's genome.

  12. Physical Mapping • The physical map is representation of the chromosomes, providing the physical distance between landmarks on the chromosome, ideally measured in nucleotide bases. • Physical maps can be divided into three general types: - Chromosomal or cytogenetic maps, - Radiation hybrid (RH) maps, - Sequence maps. The ultimate physical map is the complete sequence itself.

  13. Lowest-resolution physical map is the chromosomal or cytogenetic map, which is based on the distinctive banding patterns observed by light microscopy of stained chromosomes. • Genetic linkage mapping can be used to locate genetic markers defined by traits observable only in whole organisms. • Radiation Hybrid maps are able to provide more precise information regarding the distance between markers than can a linkage map. • The physical map that provides the most detail is the sequence map.

  14. Cytogenetic Map Cytogenetic mapping refers to observing a map location in reference to a chromosomal banding pattern.

  15. Cytogenetic Map These methods allow a rough determination of location, but to not yield a direct measure of distance.

  16. Radiation Hybrid Mapping Radiation hybrid mapping is a method for high-resolution mapping.

  17. This data is generally displayed as a vector of numbers or letters… • 1 or + for retention • 0 or - for non-retention • 2 or ? for ambiguous or unknown • Ex. • RN_ALB 0100110102010001100100100000102210010.. • RN_HEM 0101110102000100101100200010100110010

  18. Sequence Maps • 95% of genecontaining part of human sequence finished to 99.99% accuracy • Sequence maps show genetic markers, as well as the sequence between the markers, measured in base pairs.

  19. Common Sources of Sequence Tagged Sites • Expressed Sequence Tags (ESTs): short sequences obtained by analysis of complementary DNA (cDNA) clones. • Simple Sequence Length Polymorphisms (SSLPs): arrays of repeat sequences that display length variations. SSLPs that are polymorphic and have already been mapped by linkage analysis are particularly valuable because they provide a connection between genetic and physical maps. • Random Genomic Sequences: they are obtained by sequencing random pieces of cloned genomic DNA or by examining sequences already deposited in a database.

  20. References • http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/ApplMapping.shtml • http://www.ncbi.nlm.nih.gov/About/primer/mapping.html • http://www.genome.gov/10000715 • http://www.ornl.gov/sci/techresources/Human_Genome/medicine/medicine.shtml • http://www.animalgenome.org/edu/gene/index.html • Mapping and Sequencing the Human Genome: Science, Ethics, and Public Policy. 1992. BSCS and American Medical Association, Colorado Springs, CO., p. 33. • Bio_Primer_Text_v_0_14_3_1 • http://westnilevirus.okstate.edu/research/2004rr/13/13.htm • And ………some downloaded ppt.

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