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PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
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PCR

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  • 1. PCR, Sanger Sequencing, and ABCA3: Principles and Practice Daniel Diner Very Nice!Thursday, August 2, 12
  • 2. PCR: Principles Exponentially amplifies small bits of DNA strands Regions usually consist of <10 kb, but some machines allow fragments of up to 40kb Relies on thermal cycling Quick and inexpensive Generates thousands to millions of copiesThursday, August 2, 12
  • 3. PCR: A History First proposed by Kjell Kleppe and Har Gobind Khorana (Nobel Laureate, 1968) in a 1971 paper to the Journal of Molecular Biology Modern method patented by, and generally credited to Kary Mullis in 1983 Submitted to Scientific American in 1990: “Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat” Won Nobel Prize in Chemistry and Japan Prize in 1993Thursday, August 2, 12
  • 4. PCR: Procedure 1. Denaturing Step: Short heating that disrupts base hydrogen bonds, yielding single- stranded moleculesThursday, August 2, 12
  • 5. PCR: Procedure 2. Annealing Step: Temperature cools, allowing single strands to bond with complementary primersThursday, August 2, 12
  • 6. PCR: Procedure 3. Extension/Elongation: Temperature raised to optimize polymerase activity, polymerase adds complementary dNTPs to template, thus synthesizing new DNAThursday, August 2, 12
  • 7. PCR: Procedure RepeatThursday, August 2, 12
  • 8. Sanger Sequencing: Principles Developed in 1977 by Fredrick Sanger et. al Sanger shared 1980 Nobel Prize with an American team that developed a similar protocol Permits us to read nucleotide sequences and search for mutations Crucial tool for genetic disorder diagnosisThursday, August 2, 12
  • 9. Sequencing PCR results in series of DNA fragments consisting of different lengths DNA is denatured and run through electrophoresis Differently-sized bands are separated from one another Fragment terminators are read: each nucleotide gives off distinct wavelength Aggregate data gets us the complete segment sequenceThursday, August 2, 12
  • 10. Genes Cole lab researches role of ABCA3, SFTPB, SFTPC, and NKX2-1 in surfactant dysfunction/deficiency in newborns Summer students worked primarily with ABCA3 Encodes protein that is member of the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranesThursday, August 2, 12
  • 11. Referrals Cole lab receives DNA samples from referral patients from across the globe We provide free sequencing in exchange for permission to add patient sequences to database: builds rich sequence collection of otherwise rare disorders Summer students sequenced ABCA3 (32 exomes) of referral samples (13 in total)Thursday, August 2, 12
  • 12. I identified a patient heterozygous for two mutations c.589A>C or T197P (protein change) c.3863-98C>T (splicesite mutation) Each mutation came from one parentThursday, August 2, 12

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