Pcr, shotgun and microarray

1. PCR
The Polymerase Chain Reaction (PCR) provides an extremely sensitive
means of amplifying relatively large quantities of DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq polymerase, the
DNA polymerase that is used by the bacterium Thermus aquaticus that was
discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
PCR
DNA sequencing
Microarrays
Mass-spec

2.  The Polymerase Chain Reaction (PCR) provides an extremely sensitive means
of amplifying relatively large quantities of DNA

 First described in 1985, Nobel Prize for Kary Mullis in 1993
 The technique was made possible by the discovery of Taq polymerase, the DNA
polymerase that is used by the bacterium Thermus aquaticus that was
discovered in hot springs
 The primary materials, or reagents, used in PCR are:
 - DNA nucleotides, the building blocks for the new DNA
 - Template DNA, the DNA sequence that you want to amplify
 - Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
 - DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA

3. PCR
The cycling reactions :
There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an
automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time.
Denaturation at around 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic
reactions stop (for example the extension from a previous cycle).
Annealing at around 54°C :
Hydrogen bonds are constantly formed and broken between the single stranded primer and the
single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong
that the primer stays attached
Extension at around 72°C :
The bases (complementary to the template) are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added
complementary to the template)
PCR
DNA sequencing
Microarrays
Mass-spec

4. PCR
The different steps of PCR
PCR
DNA sequencing
Microarrays
Mass-spec

5. PCR
Exponential increase of the number of copies during PCR
PCR
DNA sequencing
Microarrays
Mass-spec

6.  Every cycle results in a doubling of the number of strands
DNA present
 After the first few cycles, most of the product DNA strands
made are the same length as the distance between the
primers
 The result is a dramatic amplification of a the DNA that
exists between the primers. The amount of amplification is
2 raised to the n power; n represents the number of cycles
that are performed. After 20 cycles, this would give
approximately 1 million fold amplification. After 40 cycles
the amplification would be 1 x 1012

7. Verification of PCR product
PCR
DNA sequencing
Microarrays
Mass-spec

8.  The most important consideration in PCR is contamination
 Even the smallest contamination with DNA could affect amplification
 For example, if a technician in a crime lab set up a test reaction (with
blood from the crime scene) after setting up a positive control reaction
(with blood from the suspect) cross contamination between the
samples could result in an erroneous incrimination, even if the
technician changed pipette tips between samples. A few blood cells
could volitilize in the pipette, stick to the plastic of the pipette, and
then get ejected into the test sample
 Modern labs take account of this fact and devote tremendous effort to
avoiding cross-contamination

9. Optimizing PCR protocols
While PCR is a very powerful technique, often enough it is not possible to
achieve optimum results without optimizing the protocol
Critical PCR parameters:
- Concentration of DNA template, nucleotides, divalent cations
(especially Mg2+) and polymerase
- Error rate of the polymerase (Taq, Vent exo, Pfu)
- Primer design
PCR can be very tricky
PCR
DNA sequencing
Microarrays
Mass-spec

10. Primer design
General notes on primer design in PCR
Perhaps the most critical parameter for successful PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length
- specificity and the temperature of annealing are at
least partly dependent on primer length
- oligonucleotides between 20 and 30 (50) bases are highly sequence specific
- primer length is proportional to annealing efficiency: in general, the longer the
primer, the more inefficient the annealing
- the primers should not be too short as specificity decreases
PCR
DNA sequencing
Microarrays
Mass-spec

11. DNA Sequencing
The different steps in cycle sequencing
PCR
DNA sequencing
Microarrays
Mass-spec

12. DNA Sequencing
Cycle sequencing
chain termination
Taken from:
http://www.mbio.ncsu.edu/JWB/MB409/
lecture/lecture04/lecture04.html
PCR
DNA sequencing
Microarrays
Mass-spec

13. DNA Sequencing
Sequencing systems
PCR
DNA sequencing
Microarrays
Mass-spec
LICOR DNA 4300
ABI 3100

14. DNA Sequencing
Snapshots of the
detection of the
fragments on the
sequencer
four-dye system single-dye system
PCR
DNA sequencing
Microarrays
Mass-spec

15. DNA Sequencing
Chromatogram file
PCR
DNA sequencing
Microarrays
Mass-spec

16. DNA Sequencing
The linear amplification of the gene in sequencing
PCR
DNA sequencing
Microarrays
Mass-spec

17. Massively parallel measurements of gene
expression: microarrays
• Defining the “transcriptome”
• The northern blot revisited
• Detecting expression of many genes: arrays
• A typical array experiment
• What to do with all this data?
Brown and Botstein (1999) “Exploring the new world
of the genome with DNA microarrays” Nature
Genetics 21, p. 33-37.

18. DNA
RNA
protein
genome
“transcriptome”
“proteome”
(we have this)
(we want these)

19. 1) Study all transcripts at same time
2) Transcript abundance usually correlates with level
of gene expression--much gene control is at level
of transcription
3) Changes in transcription patterns often occur as a
response to changing environment--this can be
detected with a microarray

20. Detection of mRNA transcripts
• Northern Blot -- immobilize mRNA on membrane,
detect specific sequence by hybridization with one
labeled probe--requires a separate blotting for
each probe
• DNA microarray -- immobilize many probes
(thousands) in an ordered array, hybridize (base
pair) with labelled mRNA or cDNA

21. Generating an array of probes
 Identify open reading frames (orfs)
1) PCR each orf (several for each orf), attach (spot)
each PCR product to a solid support in a specific
order (pioneered by Pat Brown’s lab, Stanford)
2) Chemically synthesize orf-specific oligonucleotide
probes directly on microchip (Affymetrix)

22. http://derisilab.ucsf.edu/microarray/
(Derisi Lab at UCSF)
The chip defines
the genes you are
measuring
The hybridization
represents the
measurement
The RNA comes
from the cells and
conditions you are
interested in

24. A print head for generating arrays of probes
Print head travels from DNA probe source
(microtiter plate) to solid support (treated glass
slide)
Small amount of DNA probe is put on a specific
spot at a specific location
Each spot (DNA probe sequence) has a specific
“address”
QuickTime™ and a
TIFF (Uncompressed) decom press or
are needed to s ee this picture.
QuickTime™ anda
TIFF ( Uncompressed) decompressor
are neededtosee this pictur e.
Print head
Printing needles

27. A yeast array experiment
vegetative sporulating
Isolate mRNA
Prepare fluorescently labeled
cDNA with two different-
colored fluors
hybridize read-out

28. Example microarray data
Green: mRNA more
abundant in vegetative
cells
Red: mRNA more
abundant in sporulating
cells
Yellow: equivalent mRNA
abundance in vegetative
and sporulating cells

29. Mass spectrometry for identifying proteins in a
mixture
From J.R. Yates 1998 “Mass spectrometry and the age of the proteome” J Mass
Spec. 33, p 1-19
Liquid chromatography and
tandem mass spectrometry
Software for processing data

30. Shotgun sequencing
 Shotgun sequencing dispenses with the need for
mapping and so is much faster. It involves chopping
the DNA into fragments of size c. 2000 base pairs
(bps) and 10000 bps, sequencing the first and last 500
bps of each fragment. It then uses computer
algorithms to assemble the entire sequence from the
sequenced fragments.

31. Speed and accuracy of sequencing
 Shotgun sequencing is much faster- it took a matter of
months to obtain a draft sequence of the fruit-fly,
Drosophila Melanogaster (135Mbps), when the
state-funded conventional sequencing effort had taken
several years to achieve a similar level of completion.
 BUT assembly of pieces, in eg. the human (3x109 bps),
requires very powerful computers
 AND repetitive DNA, which is common in eukaryotic
genomes, causes great difficulties in the assembly
process – may get it wrong.

32. Conclusions
 Sequencing DNA involves:
 Amplifying it by PCR or cloning
 Chopping it up into manageable bits
 Replicating it with fluorescently-tagged
dideoxynucleotides
 Running the different length fragments on a gel and
reading this
 Assembling the pieces (sequences of manageable bits).
 Shotgun sequencing is faster than mapping-based
assembly methods, but can have accuracy
problems.

33.  Sequence data is stored in online databases
 Extracting useful information and patterns from such
data is part of bioinformatics and often employs
intelligent systems techniques.