PCR

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PCR

  1. 1. PCR, Sanger Sequencing, and ABCA3: Principles and Practice Daniel Diner Very Nice!Thursday, August 2, 12
  2. 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. 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. 4. PCR: Procedure 1. Denaturing Step: Short heating that disrupts base hydrogen bonds, yielding single- stranded moleculesThursday, August 2, 12
  5. 5. PCR: Procedure 2. Annealing Step: Temperature cools, allowing single strands to bond with complementary primersThursday, August 2, 12
  6. 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. 7. PCR: Procedure RepeatThursday, August 2, 12
  8. 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. 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. 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. 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. 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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