PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?Continue reading “Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.”
The concept of cell death as a normal cell fate was articulated only three years after Schleiden and Schwann introduced the Cell Theory when, in 1874, Vogt described natural cell death as an integral part of toad development (as cited in Cotter and Curtin, 2003). Since these early observations, natural cell death has been described “anew” several times. In 1885 Flemming provided the first morphological description of a natural cell death process, which we now label “apoptosis”, a term coined by Kerr and colleagues to describe the unique morphology associated with a cell death that differs from necrosis (as cited in Kerr et al. 1972).
In the 1970s and 1980s, studies revealed that apoptosis not only had specific morphological characteristics but that it was also a tightly regulated process with specific biochemical characteristics. Studies of cell lineage in the nematode, Caenorhabditis elegans, showed that apoptosis was a normal feature of the nematode’s invariant developmental program (Hengartner, 1997). At the biochemical level, Wyllie showed that DNA degradation by a specific endonuclease during apoptosis resulted in a DNA ladder composed of mono- and oligonucleosomal-sized fragments (Wyllie, 1980).
These and many other studies have proven that apoptosis is a critical component of development, and when it doesn’t happen appropriately, it can be pathological, leading to cancers or other diseases. Therefore, understanding how and when apoptosis occurs and the many signals that can trigger this process is a focus of many laboratory experiments.
There are many ways to detect apoptosis in cells or tissues. This blog describes some of the most common ones. Continue reading “Detecting Apoptosis”