Investigation of the Properties of Stem Loop DNA
Primary Faculty Mentor’s Name
Angela Spencer
Proposal Track
Student
Session Format
Visual Arts
Abstract
A stem loop is a strand of DNA or RNA containing a double stranded self-complementary region (the stem) and a single stranded region (the loop). RNAs such as tRNA and ribozymes contain stem loop structures that are important for their activity. While stem loop RNAs are more commonly found in nature, stem loop DNAs are known to play a role in some aspects of DNA replication and transcription. In addition, stem loops have a variety of in vitro uses such as molecular beacons, nanothermometers and DNA aptamers.
Electrophoresis is a widely used technique that provides a simple method of separating nucleic acids based on size. Polyacrylamide gels are used for separating small DNA or RNA molecules (2-500 bp) and are run in two different formats; native or denaturing. In native polyacrylamide gels, DNA maintains any secondary structure it possesses and thus, migrates through the gel based on size and shape. On denaturing gels, secondary structure is disrupted using a denaturant such as urea, and DNA migrates through the gel based on size alone. Denaturing polyacrylamide gels commonly use urea concentrations from 6-8 M. It is known that DNAs with significant secondary structure are often difficult to denature during gel electrophoresis. In order to better understand the behavior of DNA during polyacrylamide gel electrophoresis, we designed and purchased five stem loops with 10 base pairs (bp) in the stem region with 3-30 nucleotides (nt) in the loop region, as well as 6 stem loops with 20 bp in the stem and 5-60 nt in the loop.
Based on a thorough analysis of the mobilities of our stem loop DNA constructs on both native and denaturing polyacrylamide gels, we found that many of these stem loops, specifically the stem loops with the largest stem to loop ratio, do not denature under standard denaturing polyacrylamide gel conditions. Gels containing higher concentrations of urea up to 9.5 M (the solubility limit of urea in solution) were moderately effective in denaturing stem loops that possess stable secondary structures. However, stem loops with stable conformations (ΔG < -22 kJ/mol at 25°C for 20 bp in the stem, and ΔG < -8 kJ/mol at 25 °C for 10 bp in the stem) were not denatured in gels containing 9.5 M urea.
Of additional interest in this study was to attempt to make correlations between the ability of these stem loops to denature on gels and the ability to amplify the stem loops using polymerase chain reaction (PCR). While traditional PCR parameters were unsuccessful at amplifying stem loop DNAs, high concentrations of primer as well as extended and consolidated annealing and extension temperatures allowed for successful amplification many of the stem loop DNAs. However, the DNAs with particularly stable stem loops were not able to be amplified under any conditions tested.
This deeper understanding of stem loop DNA behavior will be useful for researchers designing stem loops for in vitro technologies that employ stem loops.
Keywords
biochemistry, nucleic acids, stem loop DNA, polymerase chain reaction
Award Consideration
1
Location
Room 2905
Presentation Year
2015
Start Date
11-7-2015 1:00 PM
End Date
11-7-2015 2:00 PM
Publication Type and Release Option
Presentation (Open Access)
Recommended Citation
Baumert, Delphine, "Investigation of the Properties of Stem Loop DNA" (2015). Georgia Undergraduate Research Conference (2014-2015). 30.
https://digitalcommons.georgiasouthern.edu/gurc/2015/2015/30
Investigation of the Properties of Stem Loop DNA
Room 2905
A stem loop is a strand of DNA or RNA containing a double stranded self-complementary region (the stem) and a single stranded region (the loop). RNAs such as tRNA and ribozymes contain stem loop structures that are important for their activity. While stem loop RNAs are more commonly found in nature, stem loop DNAs are known to play a role in some aspects of DNA replication and transcription. In addition, stem loops have a variety of in vitro uses such as molecular beacons, nanothermometers and DNA aptamers.
Electrophoresis is a widely used technique that provides a simple method of separating nucleic acids based on size. Polyacrylamide gels are used for separating small DNA or RNA molecules (2-500 bp) and are run in two different formats; native or denaturing. In native polyacrylamide gels, DNA maintains any secondary structure it possesses and thus, migrates through the gel based on size and shape. On denaturing gels, secondary structure is disrupted using a denaturant such as urea, and DNA migrates through the gel based on size alone. Denaturing polyacrylamide gels commonly use urea concentrations from 6-8 M. It is known that DNAs with significant secondary structure are often difficult to denature during gel electrophoresis. In order to better understand the behavior of DNA during polyacrylamide gel electrophoresis, we designed and purchased five stem loops with 10 base pairs (bp) in the stem region with 3-30 nucleotides (nt) in the loop region, as well as 6 stem loops with 20 bp in the stem and 5-60 nt in the loop.
Based on a thorough analysis of the mobilities of our stem loop DNA constructs on both native and denaturing polyacrylamide gels, we found that many of these stem loops, specifically the stem loops with the largest stem to loop ratio, do not denature under standard denaturing polyacrylamide gel conditions. Gels containing higher concentrations of urea up to 9.5 M (the solubility limit of urea in solution) were moderately effective in denaturing stem loops that possess stable secondary structures. However, stem loops with stable conformations (ΔG < -22 kJ/mol at 25°C for 20 bp in the stem, and ΔG < -8 kJ/mol at 25 °C for 10 bp in the stem) were not denatured in gels containing 9.5 M urea.
Of additional interest in this study was to attempt to make correlations between the ability of these stem loops to denature on gels and the ability to amplify the stem loops using polymerase chain reaction (PCR). While traditional PCR parameters were unsuccessful at amplifying stem loop DNAs, high concentrations of primer as well as extended and consolidated annealing and extension temperatures allowed for successful amplification many of the stem loop DNAs. However, the DNAs with particularly stable stem loops were not able to be amplified under any conditions tested.
This deeper understanding of stem loop DNA behavior will be useful for researchers designing stem loops for in vitro technologies that employ stem loops.
