INFORME DE ACTIVIDADES CON RECICLADORES MANIZALES PARA PRESENTAR AL PROGRAMA PGIR Y ASOCIADOS ARCA.

Am plification of normal DNA

Six different m ethods w ere used to amplify genomic DNA. In the first instance DN A from pooled norm al reference DNA w as used to optim ise each m ethod before any tum our DNA w as used. Concentrations ranging from Ing, lOng, 50ng an d lOOng w ere used as a representation of the low concentrations of DNA available in some of the tu m our samples. Two m ethods, C heung (1996) and K uukasjarvi et al. (1997) d id n o t successfully am plify n o rm al D N A an d a decision was m ade not to continue the evaluation of these m ethods. The m ethods of Speicher et al. (1993), James (personal communication), Feuerstein (personal com m unication) and H uang et al. (2000) were all successful in the am plification of norm al DNA. A com parison of the fragm ent sizes produced can be seen in Figure 3.1. and Table 3.1.

1 2 3 4 5 6 7 8 9 10 11 1213 1415 16 1718 1920 2122232425

8.4kb

2.3kb

702bp

Figure 3.1: DOP-PCR amplification of normal blood DNA.

For each m ethod analysed Ing, lOng, 25ng, SOng, 75ng and lOOng starting DNA were used. Lane 1 is Lambda DNA/£co911 marker; Lanes 2 - 7 DNA amplified using Feuerstein's m ethod; Lanes 8 - 1 3 DNA amplified using James' m ethod; Lanes 14 -19 DNA am plified using H u an g 's m ethod; Lanes 20-25 DNA amplified using Speicher's m ethod.

Feuerstein James Huang Speicher

Ing <702bp <702bp - 3.6Kb <702bp - 2.3Kb <702bp - 1.2Kb lOng <702bp <702bp - 3.6Kb <702bp-2.3K b <702bp - 1.3Kb 25ng <702bp <702bp - 3.6Kb <702bp - 3.6Kb <702bp - 1.9Kb 50ng <702bp <702bp-4.3K b <702bp - 6.3Kb <702bp - 2.3Kb 75ng <702bp <702bp - 8.4Kb <702bp - 6.3Kb <702bp - 8.4Kb lOOng <702bp <702bp - 8.4Kb <702bp - 8.4Kb <702bp - 8.4Kb

Table 3.1: Com parison of fragment sized produced by four DOP-PCR methods. The am ount of starting DNA is given in the left-hand column.

Feuerstein's m ethod resulted in the smallest fragm ents of DNA being produced. Regardless of the am ount of starting DNA used, the fragm ents produced were alw ays less than 702bp in size. W ith all the other m ethods the fragm ent size increased w hen the am ount of starting DNA w as increased. The m ethods of

James and H uang both resulted in a large volum e of DNA in the 702bp - 2.3Kb size range being produced. This was seen w ith all starting concentrations using H u a n g 's m ethod, b u t only w ith starting concentrations of 50 - lOOng using Jam es' m ethod. The DNA in this size range is optim al for use in CGH. It was decided to use 50ng of DNA in all subsequent DOP-PCR experim ents, as this w as a fair representation of the samples available for analysis and p ro d u ced DOP-PCR fragm ents within the size range required for CGH.

A m plification of tum our DNA

T um our sam ples that had been analysed previously by Tracy W arr and A ndrew Ellsmore using standard CGH protocols were used as controls to determ ine the sensitivity of each DOP-PCR protocol for am plifying tu m o u r DNA. T um ours IN2809 an d IN2675, w ere used as controls in experim ents u sin g Jam es', Feuerstein's and Speicher's protocols, and IN859, IN I265 and IN2376 w ere used as controls in experim ents u sing H u a n g 's protocol. A su m m ary of the aberrations found in these tum ours can be seen in Table 3.2. Each protocol resulted in the successful amplification of each of the control DNA sam ples (data not shown).

IN Source A m plicons Gains Losses

859 CC Ip 7p 20p 14 16 17p 22q

1265 CC 3q 6q 7p 9p 4

2376 FF l q 2 7q 9p l i p 13 12q 16q

2675 FF 2q 4q 5q 6q 12q 13q 18q 16p 17 20 22q

2809 FF Ip 7q 12p Ip 2p 2q 7 8q 12p X 15 22

T able 3.2: Sum m ary of copy num ber aberrations in the tum ours used as controls for DOP-PCR experiments. CC = short term cell culture, FF = fresh frozen biopsy m aterial

L abelling of norm al DNA for use in CGH experim ents

A fter the successful optim isation of the DOP-PCR for am plification of norm al DNA, a labelling step was incorporated into the reaction in order to assess the

effects of fluorochrome incorporation on the DNA fragment size. In each instance 50ng DNA was amplified and lOmM SpectrumGreen dUTP incorporated into the DNA. N orm al DNA was labelled using F euerstein's, Jam es' and H u a n g 's m ethods. DNA am plified using Speicher's m ethod w as labelled using the labelling steps from the other three m ethods. In each instance an identical reaction w as perform ed w ith no fluorochrom e added. Figure 3.2 show s the fragm ent sizes produced using each m ethod, and Table 3.3 gives a sum m ary of the fragm ent sizes produced using each method.

1 2 3 4 5 6 7 9 10 11 12 13

1.9kb

1.2kb

702bp

Figure 3.2: Comparison of product sizes from different DOP-PCR labelling methods. Lane 1 shows the Lambda DNA/Eco911 m arker. Lanes 2 & 3 show DNA am plified and labelled using James' m ethod. Lanes 4 & 5 show DNA am plified by Speicher's m ethod and labelled using James' m ethod. Lanes 6 & 7 show DNA am plified and labelled using H u an g 's m ethod. Lanes 8 & 9 show DNA am plified using Speicher's m ethod and labelled using H uang's m ethod. Lanes 10 & 11 show DNA amplified and labelled using Feuerstein's m ethod and Lanes 12 & 13 show DNA amplified by Speicher's m ethod and labelled using Feuerstein's method.

Labelling m ethod A m plification m ethod

James

James H uang Feuerstein

702bp - 1.9Kb

H uang 702bp - 1 .3 Kb

Feuerstein 702bp - 1.1Kb

Speicher 702bp -1 .3 Kb 702bp - 1 .3 Kb 702bp - 1.9kb

T able 3.3; Sum m ary of fragm ent sizes produced d uring DOP-PCR labelling of 50ng norm al DNA. Speicher's protocol did not include a labelling step therefore DNA amplified by this m ethod was labelled using the other three m ethods.

The labelling step resulted in reduction in DNA fragm ent size to betw een 702bp and 1.9Kb. Fragm ents of this size are suitable for use in CGH experiments. The addition of the fluorochrom e resulted in fragments of equal size to the reactions w here no fluorochrome had been added.