Denaturation of DNA As DNA is heated, it reaches a temperature where the strands seperate (DNA melts). The base pairs are separated as the H-bonds between them are broken and the strand unwind. This results in single stranded DNA. It is possible to follow this process in a spectrophotometer, by observing the change in absorbance at 260nm. Unstacked bases (random orientation) absorb more light than neatly stacked (oriented) base-pairs. This results in a melting curve. The temperature at which DNA is half unfolded is called the melting temperature. Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by.... Base Composition - higher the GC content, the higher the Tm. Ionic Strength - as the ionic strength increases, so does Tm. Double helical DNA is stabilised by cations. Divalent cations (eg Mg2+) are more effective than monovalent cations (+ or K+). Organic Solvents - formamide for instance lowers the Tm by weakening the hydrophobic interactions. Reannealing of DNA If melted DNA is cooled slowly, complementary strands will pair up again. This process is called "annealing". Only complementary strands can anneal. This is an important feature of DNA which is utilised in the laboratory when carrying out DNA hybridisation. Perfect annealing requires the perfect matching of base pairs, although mis-matching occurs quite often. This gives less stable DNA, and can be monitored by watching the Tm. If the Tm lowers by 1 o, then 1% mismatch has occurred. The conditions of annealing, salt concentration and temperature, can determine the amount of mismatch that occurs. But it must be said that not all mismatching is bad. If, for instance, a gene was found in a rat, and researchers needed to know if a similiar gene was found in humans, then they would need to allow some mismatch, since the genetic sequences are unlikely to be exactly the same. "Stringency" is the term given to the conditions of annealing which control the extent of mismatching allowed. If the stringency of hybridisation is high, there is little or no mismatch with 95-100% of base-pairs matched correctly. At low stringency, only 40-50% of base-pairs are correctly repaired. You have successfully completed this set of tutorials. Congratulations, I hope you have found them interesting, and have enjoyed looking at the pictures. Good luck with your exams. If you have any comments you can e-mail me at the e-mail address below. Comments to j.tweedie@massey.ac.nz Institute of Molecular BioSciences College of Science, Massey University Private Bag 11222, Palmerston North, New Zealand last changed 18 February 1999 Copyright © 1999 Massey University
As DNA is heated, it reaches a temperature where the strands seperate (DNA melts). The base pairs are separated as the H-bonds between them are broken and the strand unwind. This results in single stranded DNA. It is possible to follow this process in a spectrophotometer, by observing the change in absorbance at 260nm. Unstacked bases (random orientation) absorb more light than neatly stacked (oriented) base-pairs. This results in a melting curve. The temperature at which DNA is half unfolded is called the melting temperature. Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by.... Base Composition - higher the GC content, the higher the Tm. Ionic Strength - as the ionic strength increases, so does Tm. Double helical DNA is stabilised by cations. Divalent cations (eg Mg2+) are more effective than monovalent cations (+ or K+). Organic Solvents - formamide for instance lowers the Tm by weakening the hydrophobic interactions. Reannealing of DNA If melted DNA is cooled slowly, complementary strands will pair up again. This process is called "annealing". Only complementary strands can anneal. This is an important feature of DNA which is utilised in the laboratory when carrying out DNA hybridisation. Perfect annealing requires the perfect matching of base pairs, although mis-matching occurs quite often. This gives less stable DNA, and can be monitored by watching the Tm. If the Tm lowers by 1 o, then 1% mismatch has occurred. The conditions of annealing, salt concentration and temperature, can determine the amount of mismatch that occurs. But it must be said that not all mismatching is bad. If, for instance, a gene was found in a rat, and researchers needed to know if a similiar gene was found in humans, then they would need to allow some mismatch, since the genetic sequences are unlikely to be exactly the same. "Stringency" is the term given to the conditions of annealing which control the extent of mismatching allowed. If the stringency of hybridisation is high, there is little or no mismatch with 95-100% of base-pairs matched correctly. At low stringency, only 40-50% of base-pairs are correctly repaired. You have successfully completed this set of tutorials. Congratulations, I hope you have found them interesting, and have enjoyed looking at the pictures. Good luck with your exams. If you have any comments you can e-mail me at the e-mail address below. Comments to j.tweedie@massey.ac.nz Institute of Molecular BioSciences College of Science, Massey University Private Bag 11222, Palmerston North, New Zealand last changed 18 February 1999 Copyright © 1999 Massey University
It is possible to follow this process in a spectrophotometer, by observing the change in absorbance at 260nm. Unstacked bases (random orientation) absorb more light than neatly stacked (oriented) base-pairs. This results in a melting curve.
Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by....
Reannealing of DNA If melted DNA is cooled slowly, complementary strands will pair up again. This process is called "annealing". Only complementary strands can anneal. This is an important feature of DNA which is utilised in the laboratory when carrying out DNA hybridisation. Perfect annealing requires the perfect matching of base pairs, although mis-matching occurs quite often. This gives less stable DNA, and can be monitored by watching the Tm. If the Tm lowers by 1 o, then 1% mismatch has occurred. The conditions of annealing, salt concentration and temperature, can determine the amount of mismatch that occurs. But it must be said that not all mismatching is bad. If, for instance, a gene was found in a rat, and researchers needed to know if a similiar gene was found in humans, then they would need to allow some mismatch, since the genetic sequences are unlikely to be exactly the same. "Stringency" is the term given to the conditions of annealing which control the extent of mismatching allowed. If the stringency of hybridisation is high, there is little or no mismatch with 95-100% of base-pairs matched correctly. At low stringency, only 40-50% of base-pairs are correctly repaired. You have successfully completed this set of tutorials. Congratulations, I hope you have found them interesting, and have enjoyed looking at the pictures. Good luck with your exams. If you have any comments you can e-mail me at the e-mail address below. Comments to j.tweedie@massey.ac.nz Institute of Molecular BioSciences College of Science, Massey University Private Bag 11222, Palmerston North, New Zealand last changed 18 February 1999 Copyright © 1999 Massey University
If melted DNA is cooled slowly, complementary strands will pair up again. This process is called "annealing". Only complementary strands can anneal. This is an important feature of DNA which is utilised in the laboratory when carrying out DNA hybridisation. Perfect annealing requires the perfect matching of base pairs, although mis-matching occurs quite often. This gives less stable DNA, and can be monitored by watching the Tm. If the Tm lowers by 1 o, then 1% mismatch has occurred. The conditions of annealing, salt concentration and temperature, can determine the amount of mismatch that occurs. But it must be said that not all mismatching is bad. If, for instance, a gene was found in a rat, and researchers needed to know if a similiar gene was found in humans, then they would need to allow some mismatch, since the genetic sequences are unlikely to be exactly the same. "Stringency" is the term given to the conditions of annealing which control the extent of mismatching allowed. If the stringency of hybridisation is high, there is little or no mismatch with 95-100% of base-pairs matched correctly. At low stringency, only 40-50% of base-pairs are correctly repaired. You have successfully completed this set of tutorials. Congratulations, I hope you have found them interesting, and have enjoyed looking at the pictures. Good luck with your exams. If you have any comments you can e-mail me at the e-mail address below. Comments to j.tweedie@massey.ac.nz Institute of Molecular BioSciences College of Science, Massey University Private Bag 11222, Palmerston North, New Zealand last changed 18 February 1999 Copyright © 1999 Massey University
Perfect annealing requires the perfect matching of base pairs, although mis-matching occurs quite often. This gives less stable DNA, and can be monitored by watching the Tm. If the Tm lowers by 1 o, then 1% mismatch has occurred.
The conditions of annealing, salt concentration and temperature, can determine the amount of mismatch that occurs. But it must be said that not all mismatching is bad. If, for instance, a gene was found in a rat, and researchers needed to know if a similiar gene was found in humans, then they would need to allow some mismatch, since the genetic sequences are unlikely to be exactly the same.
"Stringency" is the term given to the conditions of annealing which control the extent of mismatching allowed. If the stringency of hybridisation is high, there is little or no mismatch with 95-100% of base-pairs matched correctly. At low stringency, only 40-50% of base-pairs are correctly repaired.
You have successfully completed this set of tutorials. Congratulations, I hope you have found them interesting, and have enjoyed looking at the pictures. Good luck with your exams.
If you have any comments you can e-mail me at the e-mail address below.
