Biology 475 Molecular Biology Lab Seven-Nine Genomic DNA Isolation and PCR for DGGE And DGGE- Gel Pouring and Running Elizabeth Jacobsmuhlen Spring 2003

Introduction This lab used genomic DNA isolation techniques and PCR similar to those used in week one. The probes were targeted towards the 16 s rDNA bacteria sequence. However, instead of using just an agarose gel a DGGE gel will be used as well. The DGGE gel will to separate DNA based on G/C content rather than size.

	PCR Results
The upper right hand section of this gel contains my samples. Starting from the left: Lane 1: Negative Control Lane 2: No dilution Lane 3: 1:10 dilution Lane 4: 1:100 dilution

	DGGE Methods
Methods 1: This picture is of Shanna and I preparing the high gradient and low gradient solution. Each contained the appropriate high or low gradient parent solution, Dcode dye was added to the high gradient, APS (catalyst), and Temed (catalyst). These solutions will mix together as they are poured creating a high chemical concentration at the bottom and a lower concentration at the top making a gradient from high on bottom to low on top.
Methods 2: My 1:10 and 1:100 dilution samples were loaded into separate lanes in the DGGE gel after loading dye had been added.

	DGGE Results
DGGE Results: No DNA banding patterns were visible on our gel. If they had been present the banding patterns would have indicated different populations of bacteria. Each sequence separated by its G/C content.

Discussion

The results for PCR were as follows: the negative control was positive; the 1:1 dilution was negative; the 1:10 was positive; the 1:100 was positive. The negative control was positive due to contamination. The 1:1 was negative possibly due to too much product for a band to be seen.

BLAST results from lab six are a result of a DNA sequence comparison between our samples and those that are currently in the BLAST database. The DGGE procedure does not give exact sequence results. Instead it gives information about the G/C content of the DNA sequence. The DGGE gel allows the separation of the DNA sequences by their G/C content rather than their size. The chemicals in DGGE simulate melting temperatures of the DNA strands. As the strands become denatured they separate and stop moving in the gel. Sequences with a higher G/C content will move towards the bottom of the gel in the area of higher concentration. Sequences with a lower G/C content will be denatured by the chemicals at a lower concentration and therefore will stay towards the top of the gel.

DNA was not visible on our gels after they finished running. If we had bands present, those towards the bottom of the gel would have higher G/C content then those DNA fragment bands at the top of the gel. This method allows the separation of samples of 16S DNA cut to the same lengths and amplified through PCR to be loaded into one gel. The samples will then separate based on their G/C base content. DGGE allows the cloning process to be skipped. Samples separated on the DGGE gel can be extracted and have DNA sequencing performed on them.

Applications

Heyndrickx, L. et al. Simplified Strategy for Detection of Recombinant Human Immunodeficiecy Virus Type 1 Group M Isolates by gag/env Heteroduplex Mobility Assay. Journal of Virology. 2000 74(1): 363-370.

This research focused on developing a heteroduplex mobility assay (HMA) for the gag gene which can be used to identify HIV-1 as well as the recombinants and various genetic subtypes of HIV-1. When this test is used in combination with other tests it can provide a good tool for monitoring the different genetic variations in a region and can be used in labs in developing countries. Eventually the hope is that this line of research will lead to a vaccine for HIV-1.

McCaig, A.E., Glover, L. A., & Prosser, J. I. Numerical Analysis of Grassland Bacterial Community Structure Under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns. 2001. 67(10): 4554-4559.

This research compared 16S ribosomal DNA from unimproved and improved grassland soil samples. The samples were collected and then the sequence was amplified using PCR and then DGGE (Denaturing Gradient Gel Electrophoresis) banding patterns were assessed. The product from PCR also had DNA sequencing performed on them so that those results could be compared to DGGE results. Use of different primer sets could have been the cause of different results between DGGE and sequencing results between samples collected at the same location. Lower resolution of the DGGE method was detected using the procedures listed in this article