Zhenshan WangPostdoc 2001 - 2003I got my Ph.D degree from China Agricultural University. My previous experiences were mainly trained on plant molecular genetics, especially molecular markers, such as RAPD, RFLP, AFLP and microsatellite markers. I was also trained for rice genomic physical mapping by screening BAC library. In the Edwards Lab I focused on evolutionary genomics, with house finches as experimental materials. The house finch is a common backyard bird throughout most of the contiguous United States and southeast Canada. Throughout its range, its fondness for feeding stations and for nesting conspicuously around buildings make the house finch among North America's most familiar birds. Several distinctive populations exist within the native range of the house finch. However, the evolutionary relationships among these groups needs further study. |
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In house finches, the disease caused by Mycoplasm gallisepticum (MG) is manifest as an respiratory and eye infection(conjunctivitis). House finches naturally infected with MG and held in captivity with ad libitum food developed conjunctivitis within 2-4 weeks, lost weight rapidly, and many or all died. From its initial appearance in the Middle Atlantic States, MG has spread through the entire population of house finches in the eastern U.S. and southeastern Canada. The amplified fragment length polymorphism(AFLP) technique is based on the double amplification via PCR of a subset of restriction fragments from a total digest of genomic DNA. DNA is cut with two enzymes (a rare and a frequent cutter according to their restriction site). |
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| Each fragment is ligated to adapters (not recreating the restriction site) that serve as a binding site for primers with one additional selective nucleotide included at the 3' end, which probes the internal sequence of the fragment. The selected fragments are amplified in a first reaction (pre-selective). The PCR product is then used as the template for a second amplification (selective) using primers with three additional selective nucleotides included at the 3' end. Subsets of fragments possessing the complementary sequence to the primer elongation are amplified This limits the number of scorable markers on the gel. The result is a multilocus fingerprint-like pattern, which can be scored on an automated sequencer by the use of fluorescent primers. |
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AFLP is a very reproducible technique with a high multiplex ratio,
meaning that a large number of markers can be generated in a single
reaction. By using different combinations of selective primers, an
almost unlimited number of markers can be obtained. The high multiplex
ratio and the fact that no sequence information is needed represent
big advantages over other molecular marker methods.
We employed the AFLP approach to compare genetic profiles of pre- and post-epidemic finch populations, and analysis the genetic diversity of house finches on North American continent. Subtractive hybridization is a powerful technique, which compare two populations of mRNA and helps obtain clones that are expressed in one population but not in the other. Although traditional subtractive hybridization methods have been successful in some case, they require several rounds of hybridization and are not well suited to the identification of rare messages. A new PCR-based cDNA subtraction method, termed suppression subtractive hybridization (SSH), was first developed by Siebert and colleagues. SSH is used to selectively amplify target cDNA fragments (differentially expressed) and simultaneously suppress no target DNA amplification. The method is based on the suppression PCR effect: long inverted terminal repeats when attached to DNA fragments can selectively suppress amplification of undesirable sequence in PCR procedures. SSH overcomes the problem of differences in mRNA abundance by incorporating a hybridization step that normalizes (equalizes) sequence abundance during the course of subtraction by standard hybridization kinetics. It eliminates any intermediate steps for physical separation of ss and ds cDNAs, require only one subtractive hybridization round, and can achieve greater than 1,000-enrichment for differentially expressed cDNAs. SSH is a highly effective method for generating subtracted cDNA libraries. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs and simplifies the analysis of the subtracted cDNA libraries. Microarrays containing cDNA clones have been used to compare patterns of gene expression in which thousands of genes can be examined in a single hybridization. The emerging technology of cDNA microarry hybridization offers the possibility for providing a rapid, high throughout method to screen an SSH cDNA library. We will incorporate SSH, cDNA microarry and cDNA-AFLP approaches to identify “candidate genes” or markers related to MG disease in house finches. Zhenshan Wang Dept.of Zoology University of Washington Seattle, WA 98195 Phone: 206-616-2364 Email: Zhenshan(at)u.washington.edu |
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