Competencia como propósito general

After electrophoresis, gels are stained with 0.5µg ml−1 ethidium bromide prepared in a fresh buffer for approximately one hour, and then de-stained in fresh buffer on a shaking platform. A photograph is taken (see figure4.1) for record keeping with a digital camera (Cohu) on a UV trans-illuminator, and the image acquired is stored as a 16 bit TIFF file.

Gels are then cut into sections according to sample type (unirradiated controls, 10 Gy, 50 Gy, repair for one hour, etc) vertically, along the direction of migration, and across the lanes, according to the position of the molecular weight markers, thus defining molecu- lar weight regions in which measurements of DNA content are to be carried out. Each

individual gel section is put in a plastic vial for scintillation counting (Beckman). Before scintillation fluid is added to each vial, the gel sections are melted so to decrease the degree of sample quenching, 200µl of 1 M HCl are added to each vial, and the agarose sections are melted for two hours in an oven at 95◦C . Once cooled, the agarose remains in liquid form since the presence of HCl inhibits re-polymerisation. 10 ml of scintillation fluid (Beckman Ready Safe) are added to the vials and scintillation counting is performed using a Beckman LS 6500 instrument. Some agarose gel sections from lanes that have not been loaded with 14C -labelled DNA are counted to monitor background activities. These sections have similar sizes to the other sections and are treated as mentioned above. The activities expressed in counts per minute (cpm) averaged over several non- radioactive samples, per experiment, are then used as the cpm background, to be auto- matically subtracted by the scintillation counter from the cpm of the radioactive samples. For quantification of DSBs with all the methods of analysis employed in this project (chapter3), the dpm signal measured in each gel segment is first transformed into frac- tion of total activity detected, Fi. This is obtained by dividing the number of dpm counted in a given section by the total number of dpm in the gel lane to which the section belongs, as in eq.2.3 Fi= dpm(sectioni) P jdpm(sectionj) (2.3) Assuming that [2-14C ]-thymidine is uniformly incorporated throughout the DNA, Fi also measures of the fraction of DNA mass present in section i, characterised by an aver- age molecular weight Mi = M and a molecular weight range ∆Mi. It can be easily demonstrated that Fihas the following approximate relationship withMand∆Mi:

Fi'

n Mi

×Mi×∆Mi

H (2.4)

where H in eq. 2.4 is the size of the diploid human genome (in this project this is known to be 6.4 Gbp,International Human Genome Sequencing Consortium,2001) and n(Mi)is the frequency of DNA fragments, normalised to the width of the interval∆Mi. The goodness of the approximation in eq. 2.4depends on the validity of the inequality

∆Mi/Mi 1(calculations not shown) which translates to the need to cut as many thin gel sections as possible. On the other hand, thin gel sections contain less amounts of DNA, which limits detection as described above, so a compromise is required.

The experimental frequencynof fragments is a very useful quantity that can be used to make comparisons with continuous frequency distributions. This can be related to Fi using eq.2.4, as shown in eq.2.5a.

tuitive quantity which can also be estimated using eq.2.4, as shown in eq.2.5b, where N Mi =n Mi ×∆Mi. ni ≡n Mi ' H Fi Mi∆Mi (2.5a) Ni ≡ N Mi ' H Fi Mi (2.5b) The advantage of Ni over ni is that by summing all the Ni values available one has a direct quantification of DSBs in the experimental region available (see for example

H ¨oglund et al.,2000;H ¨oglund and Stenerl ¨ow,2001;Pinto et al.,2002). This very simple

method of DSB quantification is described in §3.2.2.1. The disadvantage ofNi is that its value strongly depends on the width of the gel section, since the larger the section the greater the number of fragments. To make data more consistent one should try to combine gel sections together, so to have comparable numbers of DSBs in different sections (as shown inStenerlow and Hoglund,2002).

Other useful quantities may be defined for analysis of PFGE data. Q values are obtained from integration of the fragment size mass distribution over varying windows, delimited by an upper and a lower limit. (Cedervall and Kallman,1994). Similarly, the FAR is defined as the fraction of total DNA mass below a certain threshold sizek, which may either be the exclusion size of the gel, defined as the molecular weight size above which fragments remained trapped in the wells, or the size of one of the markers that are run with the other samples. IfMmax(j)is the largest fragment in sectionj, the FARkmay be written as the sum of all the Fj that contain fragments not larger thank, as specified in eq.2.6.

FARk =

X

j:Mmax(j)<k

Chapter 3

DNA Fragmentation and DSB rejoining

kinetics models

3.1

Introduction

Mathematical models have provided invaluable tools in this project for qualitative and quantitative analysis of the experimental data that have been obtained as part of the project. In this chapter, some of the existing models of DNA fragmentation and DSB re- joining kinetics are briefly presented. It is shown how new models have been developed in this project from a knowledge of the limitations of previously utilised models available in the literature. The design of the new models will be described in detail along with their development with the aid of diagrams, while their application to experimental data can be found in chapters4and5.