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 copies
Thursday, 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
1993
Thursday, August 2, 12

4. PCR: Procedure
1. Denaturing Step: Short
heating that disrupts base
hydrogen bonds, yielding single-
stranded molecules
Thursday, August 2, 12

5. PCR: Procedure
2. Annealing Step: Temperature
cools, allowing single strands to
bond with complementary
primers
Thursday, August 2, 12

6. PCR: Procedure
3. Extension/Elongation:
Temperature raised to optimize
polymerase activity, polymerase
adds complementary dNTPs to
template, thus synthesizing new
DNA
Thursday, August 2, 12

7. PCR: Procedure
Repeat
Thursday, 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 diagnosis
Thursday, 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
sequence
Thursday, 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 membranes
Thursday, 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 parent
Thursday, August 2, 12