Lab 6 Background

Screening Yeast Colonies

After transformation, screening for cells that have taken up the inserted DNA (often simply called an insert) are often performed. Screening is necessary because cells may or may not be transformed with all the DNA fragments and may or may not assemble the DNA fragments correctly.

The plated yeast cells grow in the unit of colonies on a plate (Fig. 6.1). Each colony is assumed to have originated from a single yeast cell (remember, yeast cells can reproduce asexually through budding). Hence, all yeast cells of a single colony contain the same genetic information. We therefore can screen each colony for its genetic makeup, in this case, whether the T7 phage genome DNA is present or absent from the yeast colony.

There are many ways to screen yeast colonies. The method used in this lab, which is the most common method, is the colony PCR screening method. The key steps to perform colony PCR are to design insert-specific primers to detect the presence of your insert, to set up and run a PCR reaction using the yeast cell genomic DNA and to visualize and analyze PCR result via gel electrophoresis.

The most important step of this method is primer design. Since the goal of the colony PCR is to detect the presence or absence of the insert, the primers must be designed such that only the presence of the insert can yield a band or bands. For example, to detect if fragment 1 and fragment 2 are correctly assembled, a primer set can be designed to amplify the fragment 1/fragment 2 sequence junction (Figure 6.2). If fragment 1 and 2 are correctly assembled, then a product will be yield (Figure 6.2, left panel); if fragment 1 and 2 are not assembled correctly (or if the yeast clone only has fragment 1 or fragment 2), then no band will be yielded after the PCR reaction (Figure 6.2, right panel).

Since we transformed the yeast with multiple DNA fragments, multiple amplifications will be done simultaneously to detect the presence of all fragment junctions in the T7 genome, namely, the F1-F2 junction, the F2-F3 junction, the F3-F4 junction and the F4-F5 junction. To do this, we will use multiple primer sets in one PCR reaction, which will generate 4 bands if all the fragments are present in the yeast. Because multiple bands are generated simultaneously using multiple sets of primers, this type of PCR is also called multiplex PCR.

Multiplex PCR is different from regular PCR in that multiplex PCR contains multiple sets of primers per reaction while regular PCR has only one set of primers per reaction. While multiplex PCR is more convenient, it is not always achievable since all primers used in a multiplex PCR need to be compatible and the resulted PCR products have to be similar in length. Therefore, multiplex PCR is not often used.