BME 210 Lecture 14 DNA Sequencing

BME 210 Lecture 14 DNA Sequencing - 14. DNA sequencing...

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14. DNA sequencing
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Structure of Biomolecules Two types of structural studies of biomolecules: 1. Determination of the monomer sequence 2. Determination of their 3D structure For a DNA, the most important aspect is its nucleotide sequence (the 3D structure doesn’t vary much) For a protein, both the amino acid sequence and the 3D structure are important
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Sequencing of short DNA Sanger method DNA sequencing - determination of the precise sequence of nucleotides (A, G, C, T) in a DNA molecule Currently, the most common sequencing method is the Sanger method. It is based on: 1. DNA replication in vitro 2. Polyacrylamide gel electrophoresis 3. Use of replication-terminating nucleotides – di-deoxynucleotides Developed by Frederick Sanger in 1970s. Nobel prize in 1980
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DNA Replication in vivo Main steps: • Unwinding of double helix by helicases (1) forming a replication bubble with two forks • Synthesis of RNA primers by primases (4) - this is because DNA polymerase can’t attach to the DNA . One primer for the leading strand, multiple primers for the lagging strand • Attachment of DNA polymerase to the RNA primers and synthesis of DNA fragments in 5`-3` direction (4) • Removal of the RNA primers; filling the gaps by DNA polymerase; stitching DNA fragments together by DNA ligase (5) Replication - the process of duplication of DNA molecule using its strands as templates replication fork
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Di-deoxynucleotides • Modified nucleotides (ddATP, ddTTP, ddCTP, ddGTP) in which di-deoxyribose contains H instead of OH at 3`position • The phosphate group of another nucleotide can’t bind here • A ddNT can attach to a growing DNA at its phosphate group but, once it is added, the next NT can’t be attached DNA replication stops Deoxynucleotide (dNT) Di-deoxynucleotide (ddNT) • Standard nucleotides (dATP, dTTP, dCTP, dGTP) in which deoxyribose contains hydroxyl ( OH ) group at 3` position • During replication, this OH group binds with phosphate group of another nucleotide elongating the DNA
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Reaction components 1. Multiple copies of template DNA (obtained by cloning or by PCR) 2. DNA polymerase 3. Replication primers (required for DNA polymerase to start synthesis) 4. Deoxynucleotides (dNTs): dATP, dTTP, dCTP, dGTP 5. Di-deoxynucleotides (ddNTs): ddATP, ddTTP, ddCTP, ddGTP
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Sanger method 1. DNA Replication • The unknown DNA is denatured and divided into 4 separate tubes Primers are added in all tubes and annealed to the template ssDNA dNTs of all 4 types are added in each tube ddNT of only one type is added in each tube at a concentration of ≈1% of the dNT concentration • DNA polymerase is added in all tubes - DNA replication runs in all tubes in parallel • Most often, complementary dNTs are added to the growing DNA chain. Occasionally, a ddNT is added
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This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 14 DNA Sequencing - 14. DNA sequencing...

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