The Quality Control And Parameter Establishment Of Nucleic Acid Preparation Under Different Conditions

In some fields of current genetic research such as forensic analysis, prenatal or preimplantation diagnosis, or oncogenetics, DNA analysis has to be performed on small pools of cells or even single cells. The limited amount of genomic DNA (gDNA) which is the so-called low copy number DNA samples often fall below the sensitivity limitations of routine DNA analysis methods. Furthermore, we can't acquire high quality samples which may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. So, the quantity and quality of genomic DNA are the restrictive factors in these tests.Formalin fixed, paraffin embedded (FFPE) tissue is the most widely available material ofor retrospective clinical studies. However, the fixationof tissue samples in formaldehyde leads to extensive crosslinking of all tissue components. As a consequence of this crosslinking, the nucleic acids isolated from these specimens are highly fragmented. The average fragment length is around 60-200bp. Degradation of DNA can occur at various stages of tissue preparation, fixation and paraffin -embedded, and these will have adverse effect on the sensitivity of DNA analysis. As a conclusion, longer than 300bp amplicons are not suitable for gene expression studies in FFPE tissue. Shorter amplicon size gave promising results for molecular purposes. But, when amplicon size is very small, it is often difficult to design primers in restricted regions, and the changes of some important gene that related with disease would be ignored.So, how to elevate the detect sensitivity of high degraded samples is a problem the researcher confront. The development and application of polymerase chain reation (PCR) made the DNA analysis on single cell be possible. It is greatly accelerate the development of forensic, molecule diagnostics and molecule pathology. But the amount of DNA in many samples is too little to detected even use the extraordinary sensitive PCR. To overcome this difficulty, several methods of whole genome amplification (WGA) has been developed, providing sufficient template for numerous genetic analyses. One approach is degenerate ologonueleotide primed PCR (DOP-PCR). DOP primers have definedsequences at the 5' end and the 3' end. Between these regions is a random hexamer sequence. PCR is performed under low-stringency conditions during the first 5 cycles followed by 35 cycles with a more stringent annealing temperature.Futhermore, the purification of DNA from a variety of samples is still a rate-limiting step in obtaining useful genotypes. Purification methods commonly used, such as phenol:chloroform extraction, use hazardous organic chemicals, require multiple centrifugations, may result in significant loss of material and can introduce amplification inhibitors. Chelex extraction is rapid but frequently leaves amplification inhibitors. Purification with silica matrices are convenient but tend to give lower yields and require extensive washing to remove the lysis buffer. The DNA IQ? System uses a novel paramagnetic resin for DNA isolation. Using the DNA IQ? System to process small casework samples requires two steps. The first step provides an easy, rapid, efficient and almost universal stain extraction method. The second step uses the paramagnetic resin to purify DNA. This system is designed to rapidly purify small quantities of DNA and give consistent yields for a specific sample type.Mitochondrial DNA (mtDNA) polymorphism has been widely used for individual/species identification and maternity testing in forensic medicine and criminal investigations, due to its unique characteristics ofmaternal inheritance, high copy number per cell, high mutation rate, and absence of recombination. Commonly, many mtDNA copies exist in a cell, is about 1000 times more than nucleus DNA. Consequently, detect mtDNA woule acquire more useful results for the samples which nucleus DNA was serious degraded.Therefor, we first establish reliable standards using cell line, including autosomal and gonosomal STR typing as well as mtDNA sequencing. Using phenol:chloroform extraction, DOP-PCR, DNA IQ^TM System and combine DNA IQ^TM System with DOP-PCR to verify the least amount cell we can amplify. According this standard to detect the quality of DNA of formalin fixed and paraffin embedded tissue.Materials and methods 1 Materials(1) cultured cell line: select the human embryo amnionic fibroblast cell(FL) and human colorectal cancer cell line RKO, the culture medium is MEM and RPMI 1640 (including 10% super calf serum respectively), cultured in Water Jacketed CO2 Incubater which have 5% CO2, temperature is 37°C. After cell counted, to adopt a centain number cells for the next experiment.(2) formalin fixed and paraffin embedded tissue: the specimen wereobtained from our affiliated hospitals and other local hospitals from 1997 to 2005. All specimens were fixed in 10% formalin and thenembedded in paraffin. 2 Methods(1) DNA from cell was prepared by phenol-chloroform extractionmethod. Dilute pro rata to confirm the least amount DNA can sucessufully amplify.(2) Select the autosomal, gonosomal STR as well as mtDNA primers. Including β2 microglobulin, Cyclin D1, D2S1338, D13S317, DXS6803, DXS7130, DYS19,DYS391, D2, D4, HVI, and HVII.(3) Using phenol-chloroform extraction method, DNA IQ^TM System, DOP-PCR, and combine DNA IQ^TM System with DOP-PCR separately to process DNA detectable to verify the least number of cells can successfully amplification.Results1. Using phenol-chloroform extraction method, the least amount of DNA that can successfully amplification was 40ng using β2 microglobulin primers.2. Using phenol-chloroform extraction method, the least number of cells that can successfully amplification was 2500.3. Using DOP-PCR, the least number was 300, the primers were β2microglobulin, D2 and D4.4. DNA IQ^TM System extraction method allowed successful amplification of β2-microglobulin fragment in amplified DNA of 125 cells, whereas the amplification ability can increase to 60 cells using primers D2, D4. But the sensitivity of primers DXS6803 and D2S1338 was 250 and 1000 cells respective.5. Combine the DNA IQ^TM System extraction method with DOP-PCR, the sensitivity of primers DX7130 was improved to 75 cells.6. In FFPE sample, the least amount of DNA that can successfully amplification was 100ng using β2 microglobulin primers. The sensitivity was 800ng using DNA IQ^TM System. Combine the DNA IQ^TM System extraction method with DOP-PCR, the amplification efficiency was 250.Conclusions1. In our laboratory, the amplification sensitivity of using DNA IQ^TM System extraction method was better than DOP-PCR.2. Combine the DNA IQ^TMSystem extraction method with DOP-PCR, the sensitivity of DNA analysis was improved to more than double times.3. Analyse the mitochondrial DNA can acquire effective results toward high degraded DNA samples.4. The DNA in the pathological samples was not only affected by the DNA degradation in the processing procedure, the prior factor may be the failing amplification because of the DNA was covered due to the different ingredients.