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.

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

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

Copies of DNA=2N

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.

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

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.

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

Real Time PCR

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

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

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.

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.

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

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…

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

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

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

Affymetrix Gene Chip

Affymetrix Expression Arrays http://www.affymetrix.com/technology/ge_analysis/index.affx

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.

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

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):13784-9. 37