Forensic analysis and law enforcement officers have many tools at their disposal to help solve crimes, including fingerprint analysis, ballistics, DNA typing and, more recently, forensic phenotyping of simple physical traits such as human eye and hair color. The forensic toolbox is ever evolving and capitalizing on new discoveries in genetics and molecular biology, allowing analysts to gather more information from a biological sample left at a crime scene and ultimately increasing our chance of catching criminals. What are some of the new technologies that we can expect to see in the not-too-distant future?
If, like me, you sometimes need more motivation to exercise consistently—even though you know that it is good for you—you may be interested in the findings of a paper published recently in PLOS Genetics. The paper showed that consistent exercise over a 6-month period caused potentially beneficial changes in gene expression. In short, regular exercise caused expression of some “good” genes, and repression of “bad” ones, and these changes appeared to be controlled by epigenetic mechanisms.
Epigenetic changes are modifications to DNA that affect gene expression but don’t alter the underlying sequence. Perhaps the best understood example of an epigenetic change is DNA methylation—where methyl groups bind to the DNA at specific sites and alter expression, often by preventing transcription. Epigenetic changes have been shown to occur throughout all stages of development and in response to environmental factors such as diet, toxin exposure, or stress. The study of epigenetics is revealing more and more about how the information stored in our DNA is expressed in different tissues at different times and under different environmental circumstances. Continue reading “Epigenetics and Exercise”
When Aristotle compared epigenetics to a net (1), he could not have predicted how right he was. Recent research has revealed that mechanisms underlying epigenetic effects are numerous and interdependent as are the knots in a net. Each epigenetic mechanism has its players: enzymes, functional groups, substrates etc. The most important aspect of an epigenetic trait is its reversibility. Methylation of DNA was the first epigenetic modification to be discovered, and 5-cytosine methylation was the first to be linked with gene expression status. Currently, the most popular method for measuring CpG island methylation status is a bisulfite treatment of DNA followed by PCR or sequencing.
In this week’s webinar, Promega R&D scientist, Karen Reece focused on a workflow from DNA purification to analysis. She described the best methods for DNA isolation, quantification, bisulfite conversion, PCR and sequencing. Continue reading “Optimizing a DNA Methylation Analysis Workflow”
I’ve talked quite a bit about bisulfite conversion and DNA methylation analysis in past posts. Aberrant methylation events have significant impacts in terms of incidence of cancer and development disregulation. Researchers studying DNA methylation are often working with DNA from “difficult” tissues such as formalin-fixed, paraffin embedded tissues, which characteristically yield DNA that is more fragmented than that purified from fresh tissue. Traditional methods for bisulfite conversion involve a long protocol, harsh chemicals, and generally yield highly fragmented DNA. The DNA fragmentation may significantly impact the utility of the converted DNA in downstream applications such as bisulfite-specific PCR or bisulfite sequencing.
An ideal bisulfite conversion system would allow for complete conversion of a DNA sample in a short period of time, provide high yield of DNA, minimally fragment the DNA, work on a wide range of input DNA amounts (from a wide variety of sample types), and, while we’re at it, be easy to use and to store. Whew! That’s quite the list. Continue reading “A New Edge in Bisulfite Conversion”
Earlier this week I had an opportunity to participate in a webinar given by Dr. Karen Reece, an R & D scientist here at Promega Corporation. The title of the webinar: “DNA Methylation Mechanisms and Analysis Methods to Study this Key Epigenetic Control”.
Karen, a fellow blogger here at Promega Connections, gave an excellent presentation, and as you know, epigenetics is a hot topic these days.
By definition, epigenetics is the ability of environmental factors to affect gene expression in a heritable manner. From Wikipedia: “In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence .” Continue reading “Webinar Redux: DNA Methylation Mechanisms and Analysis Methods”
Buried in the middle of the August issue of Nature Neuroscience is an article (1) by Oliveira, Hemstedt and Bading that caught my eye. It isn’t often that I see a paper about gene rescue in a neuroscience journal, especially in a study about cognitive decline.
I looked for a News and Views summary of the article, thinking that if the conclusions of the article were anything like what the title and abstract indicated, there must be an editorial summary. I wasn’t disappointed. Su and Tsai provided a nice summary of the paper and discussed some of the potential implications of the work (2). Continue reading “Hypomethylation in the Hippocampus: Can Age-Related Cognitive Decline in Mice Be Reversed by the Activity of One Gene?”
In my last entry, I gave a little summary of one of many techniques that are used to study DNA methylation patterns in a loci-specific fashion using the COBRA technique. This time, we’ll take a look at a high-throughput, genome-wide method for analyzing DNA methylation status using a next generation sequencing approache called bisulfite sequencing, or Bis-Seq. Continue reading “Bisulfite Conversion and Next Gen Sequencing”
Last time, I talked about the birth of the bisulfite conversion method for studying DNA methylation. Scientists exploited the fact that, when treated with sodium bisulfite, unmethylated cytosines undergo sulphonation and are converted to uracil. Methylated cytosines are unaffected by this treatment. This methylation-specific alteration of the genome gave scientists a way to study the methylation patterns in DNA. One specific method for studying loci-specific DNA methylation patterns post-bisulfite conversion is called Combined Bisulfite Restriction Analysis, or COBRA. This method uses bisulfite conversion, followed by methylation sensitive restriction enzyme digestion to detect changes in DNA methylation patterns. Continue reading “Bisulfite Conversion, Meet COBRA”
Since the seminal paper on bisulfite conversions is celebrating its 20th anniversary this year, I thought it would be nice to feature, over the next few weeks, how the bisulfite conversion technique came to be and the assays that have been developed around this seminal technique.
How It All Began…..
In 1970, Hayatsu et al. discovered a chemical interaction between sodium bisulfite and pyrimidines that would have a tremendous impact on how DNA methylation patterns and changes are studied. This group found that uracil, thymididine, and deoxycydidine were subjected to sulfonation at position six of their pyrimidine rings when treated with sodium bisulfite.1 This model was later extended to 5-methylcytosine (5mC) residue.2
In 1992, Frommer et al. described that the differing rates of converting 5mC to cytosine could be used to analyze DNA methylation patterns in genomic DNA.3 Briefly, DNA was purified from HeLa cells, placenta, liver, and sperm. A plasmid was constructed that contained the promoter and first exon of the human kininogen gene. This plasmid contained known methylation patterns and was used as a standard. The DNAs were linearized with either EcoRI or via fine needle shearing and then treated with sodium bisulfite. The samples were then dialyzed to remove any unreacted bisulfite. Dialyzed samples were dried down, the resuspended in buffer, treated with sodium hydroxide then ammonium acetate. The bisulfite reacted DNA was precipitated and resuspended in buffer. The purified, converted samples were then amplified with strand-specific primers for the human kininogen gene. The group found that it was feasible to identify specific patterns of cytosine methylation by using the discrimination in deamination reactivity of cytosine and 5-methylcytosine by bisulfite conversion.3
Now that you know how the bisulfite movement was born, we will dive into techniques to exploit this reaction. Stay tuned!
- Hayatsu, H. et al. 1970. Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry. 9, 2858–65.
- Wang, Y. et al. 1980. Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8, 4777–90.
- Frommer, M. et al. 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strand. Proc. Natl. Acad. Sci. U.S.A. 89, 1827–31.
Adversity and stress are known risk factors for psychiatric disorders, cardiovascular and immune disease, cognitive decline and other health problems. The long-term negative effects of adversity seem to be greatest if the traumatic events were experienced during childhood, when the brain and other biological systems are developing and maturing. Researchers are working to identify the mechanisms involved and have identified telomere shortening as one possible mechanism by which adversity increases morbidity and mortality. Continue reading “The Link Between Childhood Adversity and Cellular Aging”