Thank you very much for sending me the email with papers.
Your boss let you do the job. Did you get some results by the methods in the papers?
If you can give me some suggestions (your experience), I will be very grateful.
A good guy!!!!!!!!作者: PCR 时间: 2016-4-4 21:34
补充：
Amplification of DNA from whole blood.
A method is described for the amplification by PCR of human chromosomal DNA sequences from whole blood samples. Various amounts of blood samples, with either EDTA, citrate, or heparin used as the anticoagulant, have been used to determine optimal PCR conditions for each type of sample. Up to 80% (vol/vol) of whole blood sample is tolerated in PCR with Taq polymerase. Amplification from whole blood requires the optimization of salt (K+ and Mg++) according to sample volume and type of anticoagulant used. Pretreatment of fresh blood samples to lyse the leukocytes is required for EDTA-treated blood samples and is beneficent in PCR with heparin- and citrate-treated blood samples to obtain maximal amplicon amounts. A satisfactory method of pretreating samples is freeze/thawing. In addition, EDTA-treated blood samples require a heat treatment before PCR for maximal amplicon synthesis. It appears that purification of the DNA is not necessary for any of the whole blood samples analyzed by PCR. Results of amplification reactions from unpurified hepatitis作者: PCR 时间: 2016-4-4 21:34
FoLT PCR: a simple PCR protocol for amplifying DNA directly from whole blood.
FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involves the use of formamide (18% vol/vol) as well as reduced incubation temperatures (cycles of 85 degrees, 40 degrees, 60 degrees C). The type of anticoagulant used was critical: sodium heparin or EDTA being superior to lithium or fluoride heparin. Our studies indicate that FoLT PCR probably works by reducing the amount of protein coagulation and allowing more DNA template to be accessible for amplification. The sensitivity of FoLT PCR is such that a single copy gene from 5.5 nucleated cells in 1 microliter of whole blood can be detected.作者: PCR 时间: 2016-4-4 21:35
Various applications of direct PCR using blood samples.
Samples of blood or other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), meaning that isolation of DNA, involving multiple labor-intensive steps, is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances. Using this reagent cocktail, DNA from various targets can be efficiently amplified directly from various forms of blood samples without DNA isolation. 1. DNA sequences within the beta-globin gene could be amplified directly from human blood samples treated with various anticoagulants. Either fresh blood or blood samples stored frozen for up to 4 years could be used for PCR. 2. DNA sequences of up to 2056 bp within the beta-globin gene could be amplified directly from human blood samples. 3. Human chromosomal and mitochondrial DNA from different individuals could be amplified directly from blood samples. 4. Low titers of hepatitis B virus could be amplified directly from human blood samples. 5. DNA could be amplified directly from various target sequences using dried blood in a PCR tube or on a filter paper. 6. Transgenes could be detected directly in blood samples from transgenic mice.
Blood and other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), so that isolation of DNA is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances and makes DNA isolation from blood unnecessary for PCR. When this reagent was included in the PCR mixture, DNA fragments of the beta-globin gene could be efficiently amplified directly from human blood samples treated with various anticoagulants or PCR-inhibitory substances. We confirmed the usefulness of this cocktail by examining a large number of blood samples with various PCR primer sets. In addition to fresh blood, this method enabled PCR amplification from blood samples stored at 4 degrees C, -20 degrees C or -80 degrees C for a minimum of 1 year.作者: PCR 时间: 2016-4-4 21:35
