Simplified template preparation and improved direct sequencing using Taq polymerase.
نویسندگان
چکیده
A streamlined version of direct dideoxy sequencing is presented that includes template preparation as well as sequencing protocols. The method is used routinely to sequence double-stranded PCR products after minimal purification with one of the primers used in amplification. Either 35S or 32P labeling can be used with equally good results.
منابع مشابه
DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combinati...
متن کاملHeterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.
We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and therm...
متن کاملA simplified protocol for producing Taq DNA polymerase in biology laboratory
Background: Taq DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the PCR. It is a standard enzyme that is used in 90% of molecular biology labs today. The aim of this study was to produce Taq DNA polymerase enzyme in E. coli by a reliable, practical, simple and low cost method. Materials and Methods: In this study, the T...
متن کاملDNA sequencing using Taq polymerase.
Three DNA polymerases, namely E.coli DNA polymerase 1 (Klenow), reverse transcriptase and T7 DNA polymerase (sequenase), are commonly used for DNA sequencing by the chain termination method of Sanger and colleagues [1). However, the secondary structure of the DNA template can impede the progress of all three polymerases. I have developed a novel procedure in which the thermostable polymerase of...
متن کاملRapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures.
The polymerase chain reaction (PCR) and dideoxy DNA sequencing are frequently used techniques of molecular biology which utilise DNA polymerases. The high temperatures required for PCR necessitate a thermostable enzyme for DNA amplification, and DNA polymerase derived from the thermophilic microorganism, Thermus aquaticus, is used most commonly. The high optimal polymerisation temperature of th...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- PCR methods and applications
دوره 1 4 شماره
صفحات -
تاریخ انتشار 1992