1. Polymerase Chain Reaction (PCR)
Starting with VERY SMALL AMOUNTS OF DNA (sometimes a few molecules), one can amplify the DNA enough to detect it by electrophoresis.

2. Cycle 1 Cycle 2 Cycle 3 Denaturation Annealing Extension 5’ 3’ 5’ 3’

3. Number of DNA molecules
Rate of PCR 2n
Initial DNA 1 2 4 8
Number of DNA molecules

4. Copies of DNA=2N

5. RT-PCR
Polymerase chain reaction amplification of cDNA can also be used to detect specific transcripts in a RNA sample.
In this procedure, known as RT-PCR, reverse transcriptase is used to copy all of the mRNAs in an RNA sample into cDNA.
Usually, oligo dT molecules, that anneal to the poly A tails of the mRNA, are used as primers.
This single stranded cDNA can then be amplified by PCR using primers that anneal to a specific transcript sequence.

6. Reverse transcriptase
RT-PCR
5‘-Cap
mRNA
AAA(A)n
(dT)12~18
primer
anneal 3‘ 5‘
AAA(A)n
dNTP
Reverse transcriptase 5‘
AAA(A)n
Regular
PCR
cDNA:mRNA hybrid

7. The amplified DNA fragments that are produced can by analyzed by agarose gel electrophoresis.
The amount of amplified fragment produced is proportional to the amount of target mRNA in the original RNA sample.
Although less quantitative than Northern blots, RT-PCR is extremely sensitive and can be used to detect very rare mRNA species.

9. How do we accurately quantify the amount of DNA?
Real-time PCR

10. Real Time PCR

11. 3. intensifier
5. ccd detector 350,000 pixels
1. halogen tungsten lamp
2b. emission filters
2a. excitation filters
4. sample plate

12. Amplification Plot of real-time PCR
Log phase
Level off/ plateau 2 4
DNA copy number (log) 8 16 32
PCR cycle (Ct)

13. DNA sequencing
Sequencing is used to determine the precise order of nucleotides in a DNA molecule.
The Sanger di-deoxy method involves the synthesis of DNA by a DNA polymerase.
DNA synthesis is terminated at specific nucleotides by the incorporation of di-deoxy nucleotides that are missing the 3’ OH.

23. Sequence analysis
Four different reactions produce DNA fragments that are terminated (randomly) at each of the four nucleotides.
These samples are resolved by electrophoresis.
The shortest fragments, those terminated closest to the primer, run faster than the longer fragments.

25. DNA sequencing reactions can be radioactively labeled.
A C G T
DNA sequencing reactions can be radioactively labeled.
Bands detected by X-ray film exposure.
Sequence can be read in the 5’ to 3’ direction from the bottom of the image towards the top. A A T C T A A C G

26. This is great but…
Wouldn’t it be great to run everything in one lane?
Saves space and time, more efficient
Fluorescently label the ddNTPs so that they each have a different color…

27. 07_03.jpg
07_03.jpg 27

28. Automated DNA sequencers use dideoxy terminators labeled with four different fluorescent dyes.
All four reactions can be carried out simultaneously in a single reaction.

29. Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer.
The DNA sequence is read automatically.

30. Beckman CEQ 2000, 8 capillary
ABI Prism 3730, 96 capillary

31. Affymetrix Gene Chip

32. Affymetrix Expression Arrays

35. Analysis of microarrays
Microarrays allow for the simultaneous analysis of the expression of thousands of mRNAs.
Useful for determining changes in gene expression patterns from one sample tissue to another.
For example, microarrays have been used to study differences in gene expression in different tumor tissues.

36. Genes with similar expression patterns are clustered together.
Gene expression patterns can be associated with different
disease patterns.

37. Microarray example #1: Biomarker identification - lung cancer
Samples
Genes
Gene expression patterns segregate the four major morphological lung tumor subtypes
Patterns of gene expression promise to refine traditional morphologic classification of lung cancer.
Can we distinguish subsets of genes that would allow molecular-level classification of tumor subtypes?
Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24): 37