13 Genomic analysis
Many diverse studies require the determination of the abundance of large numbers of specific DNA or RNA molecules in complex mixtures, including, for example, the determination of the changes in mRNA levels of many genes. While a number of techniques have been used to estimate the relative abundance of two or more sets of mRNA, such as differential screening of cDNA libraries, subtractive hybridization, and differential display, far more superior methods have been recently developed that are particularly amenable to organisms whose entire genome sequences are known, such as S. cerevisiae. It is now practicable to investigate changes of mRNA levels of all yeast ORFs in one experiment.
Numerous companies and academic groups have developed novel approaches to DNA sample preparation, probe synthesis, target labeling and readout of arrays. The following procedures have been successfully used for determining mRNA levels in yeast: (i) the DNA Microarray System; (ii) the Oligonucleotide Microarray System; (iii) the Low-density DNA Array System; and (iv) the kRT-PCR System.
The DNA Microarray System. As a general means to address such problems as the differential expression of an entire genome, Brown (1998) and his colleagues developed a system for making microarrays of DNA samples on glass slides, probing the DNA micro-spots by hybridization with fluorescent-labeled probes, and using a laser-scanning microscope to detect the fluorescent signals corresponding to hybridization. Fluorescent labeling allows for simultaneous hybridization and separate detection of the hybridization signal from two probes, thus allowing accurate determinations of the relative abundance of specific sequences in two complex samples. For example, with the DNA Microarray System, the entire 6,400 ORFs of the yeast S. cerevisiae can be placed on one slide. The mRNA levels of all ORFs (open reading frames) can be determined after, for example, metabolic shifts or in strains deleted for a single gene (DeRisi et al., 1997).
The use of DNA Microarray System requires the following basic steps for investigating gene expression of two related cell types or conditions: (i) preparation of the large set of DNA elements, usually consisting of ORFs amplified by PCR with sets of primer pairs specific for each ORF; (ii) preparation of DNA microarrays consisting of these ORFs spotted on glass slides by a robotic printing device, the Arrayer; (iii) preparation of two related mRNAs derived from cells that differ in the trait that is to be investigated; (iv) preparation from the mRNAs of fluorescently labeled cDNA by reverse transcription in the presence of Cy3 (green) or Cy5 (red) labeled dUTP; (v) hybridization of the fluorescently labeled cDNAs to the ORFs of the DNA microarrays printed on the glass slide; and (vi) quantitative analysis of the relative abundance of the mRNAs from the degree of hybridization, using the Scanner.
The DNA Microarray System requires two machines, the Arrayer and the Scanner. The Arrayer prints DNA samples robotically onto a glass slide. After hybridization, the Scanner analyzes the two colored fluorescence of the array with a specially designed scanning confocal microscope.
The Oligonucleotide Microarray System. Modern photolithographic techniques are being used to generate miniaturized arrays of densely packed oligonucleotide probes. These oligonucleotide microarrays, or DNA chips, can then be used, for example, for comparing two sets of cDNAs, similar to the DNA Microarray System described above. Also, as with the DNA Microarray System, the hybridization pattern of fluorescently labeled cDNAs is detected by epifluorescence microscopy. This technology was developed by the Affymax Research Institute (Wodicka et al., 1997), and the oligonucleotide microarrays can be produced only by the company.
Using this technology, approximately 6,200 or almost all ORFs of the entire S. cerevisiae genome were probed for differential mRNA expression in two growth conditions (16). Approximately 12% of the ORFs, or approximately 750, showed appreciable differential expression rates in the two growth conditions.
The Low-density DNA Array System. In addition to dotting on glass at high densities, as described above for the DNA Microarray System, sets of PCR-generated ORFs can be spotted on nylon membranes, and hybridization to the dot blots can be carried out with 32P-labeled cDNA for detection of the relative levels of mRNAs. In some procedures, 1,536 dots were prepared on a single 9
x 13 cm positively charged nylon membrane. The lower density arrays on nylon membranes can be achieved by automatic robotic and hand-held devices. No special equipment is required to process the membranes once they are prepared.The kRT-PCR system. In addition to screening for the differential expression of genes by the methods described above, many investigations also require more accurate determinations of large number of mRNA levels. While in the past, Northern blot analysis has been by far the most common means to quantitate mRNA levels, new methods have been developed that are capable of producing more accurate estimations of large number of samples. Some of these are based on the automatic monitoring of reverse transcriptase initiated PCR (or kinetic monitored reverse transcriptase initiated PCR, "kRT-PCR") . Several instruments are commercial-available, such as the LightCyclerTM and the ABI PrismTM 7700 Sequence Detection System, and some are under development. The sensitivity, accuracy, and reproducibility of kRT-PCR are remarkably high, allowing the detection of 20% differences and quantitation raging from 367 to 0.00075 copies per cell of S. cerevisiae transcripts