Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites.

نویسندگان

  • Deepali Shinde
  • Yinglei Lai
  • Fengzhu Sun
  • Norman Arnheim
چکیده

During microsatellite polymerase chain reaction (PCR), insertion-deletion mutations produce stutter products differing from the original template by multiples of the repeat unit length. We analyzed the PCR slippage products of (CA)n and (A)n tracts cloned in a pUC18 vector. Repeat numbers varied from two to 14 (CA)n and four to 12 (A)n. Data was generated on approximately 10 single molecules for each clone type using two rounds of nested PCR. The size and peak areas of the products were obtained by capillary electrophoresis. A quasi- likelihood approach to the analysis of the data estimated the mutation rate/repeat/PCR cycle. The rate for (CA)n tracts was 3.6 x 10(-3) with contractions 14 times greater than expansions. For (A)n tracts the rate was 1.5 x 10(-2) and contractions outnumbered expansions by 5-fold. The threshold for detecting 'stutter' products was computed to be four repeats for (CA)n and eight repeats for (A)n or approximately 8 bp in both cases. A comparison was made between the computationally and experimentally derived threshold values. The threshold and expansion to contraction ratios are explained on the basis of the active site structure of Taq DNA polymerase and models of the energetics of slippage events, respectively.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands

Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during in vitro amplifi...

متن کامل

Correction of some genotyping errors in automated fluorescent microsatellite analysis by enzymatic removal of one base overhangs.

The conjunction of high resolution genetic maps based on (CA)n microsatellite markers (1) and fluorescent genotyping (2) has led to research programs which require the determination of hundreds of thousands of genotypes. However, the migration profile of a (CA)n microsatellite marker after PCR (polymerase chain reaction) is often complicated because of slippage of Taq polymerase during PCR, and...

متن کامل

What Is a Microsatellite: A Computational and Experimental Definition Based upon Repeat Mutational Behavior at A/T and GT/AC Repeats

Microsatellites are abundant in eukaryotic genomes and have high rates of strand slippage-induced repeat number alterations. They are popular genetic markers, and their mutations are associated with numerous neurological diseases. However, the minimal number of repeats required to constitute a microsatellite has been debated, and a definition of a microsatellite that considers its mutational be...

متن کامل

Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands

Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during in vitro amplifi...

متن کامل

Utilization of a 17 Microsatellites Set For Bovine Traceability in Czech Cattle Populations

For identification of individuals and parentage control performed by cattle breeders in the Czech Republic, a novel Finnish Bovine Genotypes™ Panel 3.1was amplified by means of one multiplex polymerase chain reaction. Bovine Panel encompasses all the 12 STR loci recommended by the International Society for Animal Genetics (ISAG) for routine use in parentage testing and identification, including...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Nucleic acids research

دوره 31 3  شماره 

صفحات  -

تاریخ انتشار 2003