Essential Biochemistry - Texas Tech University

Essential Biochemistry - Texas Tech University

Sanger or Dideoxy DNA Sequencing Components for the Chain Termination Method: DNA fragment for sequencing Primers dNTPs ddNTPs Sanger or Dideoxy DNA Sequencing What direction does the DNA polymerase elongate the DNA fragment?

Sanger or Dideoxy DNA Sequencing Migration smaller is faster through the gel (bottom smallest) Each peak represents 1 base Pyrosequencing Based on DNA synthesis Template DNA is immobilized Peak area

represents base number Pyrosequencing Potential Problem: DNA polymerase and luciferase compete for the same dATP substrate Solution: Deoxyadenosine alpha-thio

triphosphate (dATPS) is efficiently S) is efficiently used by DNA polymerase but not recognized by luciferase Pyrosequencing Requirement: Generate ATP from polymerase generated pyrophosphate Solution: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5

phosphosulfate Pyrosequencing Potential Problem: Unincorporated dNTPs can react in the next cycle producing false positives Solution: The nucleotide degrading enzyme apyrase degrades unincorporated dNTPs

between cycles Gene Amplification by Polymerase Chain Reaction (PCR) Gene specific amplification without purification Amplification via temperature cycling Special Taq (Thermus aquaticus) DNA polymerase Detection of Helicobacter pylori, Borrelia burgdorferi,

as well as hepatitis, HIV, and West Nile virus Restriction Enzyme Recognition Sites Restrictive endonucleases recognize 4-8 bp sequences within the DNA and cleave at a specific site DNA strands that are cut in a symmetric fashion are palindromic

Blunt and Sticky DNA Cuts Molecular Cloning by Recombinant DNA Technology Cut by restriction enzymes Anneal an uncatalyzed reaction Re-close by DNA ligase Screening for Colonies Containing

the Plasmid with the Insert Size-Specific Cloning Vectors Recombinant Protein Products Generating a Restriction Map Where are the restriction sites located on the 20 kb fragment that

would generate this map? . . . Transcript Analysis via Microarray/DNA Chip How to examine gene expression changes with a given treatment

mRNA cDNA fluorecent labeling cDNA/oligonucleotide Hybridization Slide scanning Data analysis Differential Arabidopsis Gene Expression

with GB03 Exposure RNA Extraction cDNA Synthesis Label Hybridize Wash Scan Block 8 Full Image

cDNA Synthesis and Labeling O HN - O O O

O P O P O P O- O- O-

O O O NH2 N OH Amino Allyl-dUTP -


Cy5 Dye N SO3- Microarray Data Analysis

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