A New Method For Identification Of Differences Between Different Genomes

The ability to identify and analyze species-specific sequences within a genome will assist in elucidating the evolutionary pattern of the species and have profound benefits for the development of evolutionary biology. Identifying the differences between the genomes of two species helps to clarify how genes are related to particular attributes, and elucidate their involvement in different biological mechanisms. For distantly related organisms, different sequences can serve as probes for intermediate species and can aid in the construction of phylogenetic trees, or can be used as a diagnostic marker for DNA-based detection. Once the genomic sequences of organisms have been established, computer-based comparisons can analyze genomic differences between species. However, with the techniques currently available, the genomes of eukaryotic organisms are too massive and complex to be sequenced efficiently without great expense. During the last few decades, various methods have been developed to identify and isolate unique sequences present in one genome but absent in another. Most of these techniques are based on the subtraction hybridization technique, which enriches the target-specific DNA fragments and removes any common sequences. An important factor in the subtraction process is the ability to determine whether the subtraction is complete. In general, high false positive efficiency results from insufficient subtraction, and yet there is currently no appropriate strategy to determine the extent of the subtraction process. Representational difference analysis (RDA) incorporated "kinetic enrichment"into PCR-based subtractive hybridization, but this method is labor-intensive and prone to high rate of false positives and negatives if not properly performed. Meanwhile, differences were mixed with the excess driver after subtraction, which increased the complexity of the template of differences, thus decreasing the amplification efficiency of the differences greatly and leading to a lower coverage, so RDA was usually applied to nearly identical genomes or differential expression. Suppression subtractive hybridization (SSH), a subtractive method based on suppression PCR, is considered the most effective way to analyze genomic differences, which has been successfully applied to analyze differences between simple genomes or differential expression. But a lack of appropriate controls precludes its use with complex genomes. Though the genomic subtraction kit from CLONTECH can control for analysis of the DNA hybridization process, the subtraction efficiency of the control in this kit does not adequately reflect the hybridization status of the samples'reaction conditions, such as the sequence complexity, concentration and quality of DNA sample. These shortcomings make it difficult to estimate the subtraction process, thus resulting in increased false positives. The inherent deficiencies of these techniques exclude them from being applied to eukaryotic organisms with large and highly variable genomes. In this report we describe a novel method to compare sequences between two complex genomes. This method utilizes a hybridization monitor system called hybridization-monitored differential analysis (HMDA), which monitors the entire hybridization process. HMDA is a PCR-based solid subtractive method, which includes the following main procedures. The two DNA samples served as driver and tester respectively. Driver DNA is immobilized on a 6 mm-diameter positively charged nylon disc, then hybridize with the adaptored tester DNA. Homologous fragments can be eliminated when bound to the driver DNA immobilized on a solid membrane, and differences are gradually enriched. After each round of hybridization, the solid membrane was removed and a new membrane immobilized the driver DNA was used for another round of subtraction. The hybridization continues until no homologous fragment could be detected by PCR. The differences between the two genomes were left in the subtraction buffer. In this research, a hybridization monitor system was established to track the entire hybridization process. 18S rDNA, which was considered as the representative of the homologous sequences of the two samples, was analyzed both during and following each round of hybridization to assess the success of the hybridization using the specific primer pairs designed based on its conserved region. If 18S rDNA was not detected in the tester DNA sample, it was hypothesized that the homologous sequences had been completely hybridized with the driver DNA immobilized on the nylon disc, and the remaining unhybridized DNAs in the hybridization buffer represented tester-specific sequences. PCR amplification was conducted using different fragments left in the hybridization buffer as template and the amplified products were then cloned into the vector. Meanwhile, To demonstrate the efficiency and feasibility of the method, we utilized the technique to analyze genomic differences between two full-sequenced yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. Other characters, such as proportion of differences in the subtracted library, coverage and enrichment efficiency were also evaluated and analyzed in this report.Partial conserved region of 18S rDNA was selected to serve as hybridization monitor sequence, which also shares over 70% homology among the fungi, humans, and plants as assessed using the BLASTN program. Specific primer pair (R1 and R2) was designed according to monitor sequence with 180bp between the primer annealing sites. The optimum annealing temperature of the primer pair was 60°C evaluated by gradient PCR. Because high ionic concentration would inhibit the activity of Taq DNA polymerase, the volume of hybridization buffer containing tester DNA added in PCR analysis was optimized. Results showed that the amplification system with a final concentration of 0.02-0.01×SSC did not affect the PCR amplification. Less than 0.2 μl of hybridization buffer (5×SSC) containing tester in 25 μl of amplification mixture was feasible to perform hybridization monitoring analysis. 18S rDNA could be detected when 100 fg DNA at least was used as template in the PCR reaction with the condition mentioned above. By using the primer pairs (R1 and R2) to other organisms, such as filamentous fungi, human, locust and citrus, the 180 bp fragment was also amplified, indicating the broad-spectrum utilization of this fragment for monitoring subtractions among eukaryotes. Because fragments left in the tester were mainly the differences after rounds of subtraction, complexity of the template for the differences decreased greatly, thus enhanced the amplification efficiency and was helpful for the increment of the coverage accordingly. Tester DNA was fully digested with the four-base-cutting restriction enzyme Tsp509Ⅰ.Digested products were mainly distributed between 200-800bp. A 60-70% of immobilization efficiency was got on nylon disc for driver DNA sheared by sonication. PCR analysis of 18S rDNA in the elution solution was performed. Results showed that no 18S rDNA was detected in the rinsing buffer after washing twice with 5×SSC for 30 min, and twice with 2×SSC for 30 min at 60°C, indicating that the membrane could be used for the following hybridization. When the hybridization of the driver and tester were present in an 800:1 ratio, the 18S rDNA gene was still detected even after four rounds of hybridization, suggesting that the hybridization was completed. Electrophoresis diagram of amplification products varied with each round of subtraction. With the smear disappearing, some bands were gradually obvious. Sequences analysis of the sequencing results showed that the size of the inserts varied from 86 to 1100 bp in length, with an average length of 410 bp. Homology search was performed by BLASTN program. Among 60 sequences, 57 (95%) had no homology in driver genome database, and 3 (5%) were false positive, which hassimilarity with the driver genome. The differential sequences were distributed across on the 13 chromosomes. Among the 52 single copy sequence, 49 (94%)were found positive differences, while 8 multicopy fragments(100%)were all different fragment when searching the tester genome database. A differential sequence, selected randomly from the subtracted library, was labeled with digoxigenin (DIG) to screen the replica filter made from the S. cerevisiae subtracted and unsubtracted plasmid libraries. Results showed that 240-fold enrichment after 4 rounds of subtraction and 4-fold enrichment per round were obtained after four rounds of subtraction. To determine the coverage, various differential sequences across the 16 chromosomes of S. cerevisiae were selected randomly from Genbank. Thirty-three primer pairs were designed based on 33 sequences different to S. pombe. PCR amplification was carried out using the final hybridization products. The coverage achieved by our method was calculated by determining the percentage of primer pairs that amplified target fragments in the subtracted tester DNA. Results showed that 26 out of 33 primer pairs were amplified target sequences in subtraction products, indicating that the coverage of the method is approximately 79%.