DNA Sequencing and Fragment Analysis

Archive for May, 2012

Epigenetics: A New Field of Genetic Research

Epigenetics is a relatively new field of research that goes beyond gene expression. It encompasses important areas of research including cell differentiation, cancer and the aging process. Despite complete sequencing of the human genome, there is still much to learn about the complexity of an individual genetic blueprint. Epigenetic research is working to better understand how cells develop into specialized tissue and perform a specified number of functions. The human begins as a single fertilized egg. The egg divides into a growing mass of identical cells. Eventually, individual groups of cells begin to look and act different than other cells. Cell groups develop into tissue and organs. How does this happen when every cell has the same genetic code?

Proximity could play a partial role in cell differentiation. Cells adhering together generally develop into the same tissue. But what boundaries separate each group?

The answers may rest in understanding the epigenome. Epigenetics is a complex field newly emerging in the genetic sciences. Although there is much to learn, there is some basic understanding summarized here.

Histones Play an Important Role in Controlling Gene Expression

DNA is a double stranded molecule that coils around into a helical formation wrapped around histone molecules. Histones are a group of proteins that condense long DNA strands. It is similar to string wrapped around a spool. How tightly DNA is wrapped around the histone molecule determines whether a particular region of DNA is available for transcription and gene expression. Tightly wound DNA is essentially hidden and this region is repressed. Other regions are loosely wound and available for transcription. Transcription is the first step in the protein production process (Figure 1).

Histone molecules are classified into one of five main groups. They are modified in the cell by chemical processes such as methylation, acetylation or other known modifications. Modification is one factor that determines whether DNA is active or repressed. Methylation of certain groups of histones allows genes to be active while others are repressed. However, external factors (like radiation) could effectively influence methylation and activate repressed genes.

Epigenetic Damage is a Cause of Cancer

Let us examine a hypothetical situation in which two individuals have identical genetic content. They are twins. Each individual has identical genes including genes that potentially cause cancer. Why does one individual develop cancer and the other does not?

Research has long understood that cancer could be caused by damage to the DNA making up the genetic blueprint. But external factors can cause damage to DNA leading to cancer as well. Factors that damage the epigenome of an individual can have the same result. An environmental factor like smoking is an example. When cells lose levels of control, they also lose specialization; they de-differentiate. Without such important controls, cells begin to divide uncontrollably into cancer. A better understanding of epigenetic damage has led to new directions in cancer treatment.

Dr. Jean-Pierre Issa explained some general aspects concerning cancer and epigenetic damage during an interview with Nova. See Nova article here.  Epigenetic damage could be related to the aging process. As an individual continues to age, cell division eventually leads to errors in the epigenome. For example, skin constantly exposed to sunlight radiation appears older than normal skin. The aging process is accelerated by stem cells dividing to repair sun damaged skin tissue. The more cells divide, the more the aging processes are accelerated. Dr. Issa utilizes this knowledge in research to determine the apparent age of certain cells.

What Affects the Epigenome?

Current research is attempting to answer this question. There are a number of known environmental conditions that cause damage to the epigenome. Radiation, tobacco and other chemicals have the potential to damage the cellular epigenome. As previously stated, the radiation from excessive sunlight can cause damage to skin tissue. Stem cells divide to repair damaged skin. Damage to skin tissue causes the need for repair and accelerates the aging of stem cells.

Epigenetics is a fascinating new field in the genetic sciences. Understanding factors that affect the epigenome has led medicine into exciting new areas of treatment. This topic is certain to be examined often in the near future.

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Primer Selection Guidelines: Good Primers Important for PCR and Automated Sequencing

DNA synthesis is the production of short, single-stranded DNA molecules (called primers or oligonucleotides) often used in the polymerase chain reaction (PCR) and DNA amplification for Sanger sequencing applications. The region of DNA amplified is determined by an exact match of the primer to its complimentary bases on a given DNA strand. Primer sequences are determined from known sequence since there must be a match to the region of DNA to be amplified.

PCR amplification requires 2 primers that determine the region of sequence amplified in the forward and reverse direction. The forward primer is designed along one strand in the direction toward the reverse primer. Likewise, the reverse primer is designed from the complimentary strand. PCR is exponential amplification in which the newly generated PCR fragment from one cycle also acts as a template for the next cycle (Figure 1A).

Amplification of DNA for Sanger sequencing differs from PCR in that a single primer is used. Amplification is the reproduction of one strand using the compliment from the original strand. Newly generated PCR fragments are single stranded and do not provide a complimentary strand that could act as a template for additional amplification (Figure 1B).

Differences in amplification between PCR and Sanger sequencing were discussed previously (Sanger Sequencing Amplification Compared to Basic PCR).

Simply designing the sequence of the primer from known sequence does not ensure the primer will anneal to the desired region and initiate amplification. The primer should be designed following a set of given standards that improve the chances of success. The guidelines for designing primers used in PCR and sequencing are fairly similar. Primers are designed in the 5’ to 3’ direction to compliment the direction of amplification.

For those utilizing PCR and Sanger sequencing in everyday applications, primer design could seem like yesterday’s news. However, we still believe reviewing good primer design guidelines is helpful to any researcher involved in genetic research.

Guidelines for Primer Design

G1. Primer length should be in the range of 18 to 22 bases. Primers less than 18 bases will have a low melting temperature (Tm values) and might not anneal to the template. There is some flexibility for designing primers longer than 18 bases. Longer primers are frequently designed from template regions that are AT-rich and need additional bases to increase the Tm value.

G2. The primer should have GC content of 50% to 55%.   This is the equivalent of 9 or 10 GC bases included in an 18 base primer. Sometimes there are regions on a template that are AT-rich which prevents meeting this guideline. In those cases it is recommended to design a primer longer than 18 bases.

G3. Primers should have a GC-lock on the 3’ end. A GC-lock is designed when 2 of the final 3 bases is a G or a C. The 3’ base should always be a G or a C.

G4. The melting temperature of any good primer should be in the range of 50OC to 55OC. However, guidelines particularly related to Tm value have some flexibility. Melting temperatures are directly related to the PCR cycle annealing temperature. Tm values that are too low may not anneal well during PCR. High values could be too stringent causing difficulty locating the correct annealing site on the template.

G5. The primer should not include poly base regions. This is when 4 or more bases in a row are the same. This guideline helps prevent potential slippage in which the primer shifts from the annealed position.

G6. Four or more bases that compliment either direction of the primer should be avoided. This prevents the primer from annealing to itself and forming what is referred to as primer-dimer. Primer-dimers have the capability of amplifying the primer itself causing short secondary sequence.

PCR Specific Guidelines

G7. Forward and reverse primers used in PCR amplification should have similar melting temperatures (+/- 2OC). This allows a 4OC difference in total melting temperatures. Researchers involved in using PCR amplification will use primer Tm values in an effort to optimize PCR cycles. Similar Tm values for forward and reverse primers aid optimization efforts. Multiplex PCR applications using multiple primer pairs should all have similar Tm values. A wide range in primer melting temperature complicates PCR optimization.

G8. Forward and reverse primers should not have regions 4 bases or longer that compliment. Just like a primer used in Sanger sequencing, forward and reverse primers used in PCR can anneal to each other and form primer-dimers.

G9. The Tm values for tailed primers should include the tail in calculating melting temperature. Yes, melting temperatures will be greater than 55OC. However, the additional bases in the tail will add to the amplified PCR fragment and become part of the priming site. Tailed primers are often used to add restriction sites to an amplified product.

Primer design is an important aspect relating to many forms of PCR including basic PCR, fragment analysis, quantitative analysis and Sanger sequencing. Below is a link that offers free primer design software…

http://frodo.wi.mit.edu/

We invite any additional guidelines or comments that may be useful to both experienced researchers and those new to primer design.

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