Transformation in Saccharomyces Cerevisiae

Transformation in Saccharomyces cerevisiae

BIOZ 310 Genetics lab

Section: L03

-ABSTRACT-

Transformation is a method that can be used to add or alter a DNA sequence in a cell. The hypothesis of the exercises that were performed was that DNA was the genetic material in Saccharomyces cerevisiae. In the experiment, an ade2::kanR DNA sequence was integrated into the DNA of budding yeast cells. This caused the cells to become resistant to kanR G418. It was also observed that cells that had integrated the DNA sequence into their DNA, had turned a shade of red after incubation as compared to the cream colored cells that did not up-take the ade2::kanR gene into their DNA. To prove that DNA was the genetic material found in budding yeast, polymerase chain reaction (PCR) was used to amplify the transformed and wild type DNA. Upon purification of the PCR products, agarose gel electrophoresis was used to determine the fragment sizes. In conjunction with restriction mapping, DNA sequence analysis, and the Basic Alignment Search Tool (BLAST), the hypothesis was thoroughly tested. As a result the fragment sizes of the transformed and the wild type cleaved at the expected sites. The transformed PCR product band size was measured to be 2,083bp, while the wild type band size measured 2,332. Both the transformed and the wild type PCR products that were treated with HindIII cleaved and created at the expected fragments. The transformed HindIII PCR product resulted in the following band sizes: 1,252bp and 831bp, while the wild type HindIII PCR products produced the following band sizes: 1,185bp, 824bp, and 323bp. A BLAST search was then used to see if there was any known DNA sequences similar to the transformed final sequence, wild type final sequence, and the kanR sequence. A match was found for each of the searches. The transformed and wild type sequences were matched to the genome of Saccharomyces cerevisiae, while the non-ADE2 sequence was matched to the...