Volume 3 no.11 November1976
Nucleic Acids Research
Sheared DNA fragment sizing: comparison of techniques
Charles P. Ordahl, Thomas R. Johnson and Arnold I. Caplan Departments of Biology and Anatomy, Case Western Reserve University, Cleveland, OH 44106, USA
Received 2 August 1976 ABSTRACT DNA fragmented by conventional French press shearing procedures (30,000 lbs/in2) has a number-average fragment size of 230 base pairs. This is considerably smaller than the 450 base pairs typically reported for DNA sheared by this method. Comparison of 5 sizing techniques indicates that sheared DNA fragment size is overestimated by either measurement of velocity sedimentation or Kleinschmidt Electron Microscopic visualization. Both adsorption grid electron microscopic visualization and gel electrophoresis yield the most reliable estimates of the mean size of small DNA fragment populations. In addition, the assessment of fragment size distribution (not possible from sedimentation analysis) potentially allows more critical evaluation of DNA hybridization and reassociation kinetic and measurement parameters. INTRODUCTION DNA reassociation and hybridization analysis requires the use of fragmented DNA Precise information regarding the size of the DNA fragments is essential to interpretation of these kinds of experiments. Fragment size is not only an integral element in the kinetics of DNA reassociation and hybridization, but also affects the kinds of DNA-DNA or DNA-RNA duplexes obtained. For some experiments it is necessary to use long (>1000 base pair) fragments of DNA. Fragments of this size have been used to determine the extent of inter4 spersion of repeated and nonrepeated sequences of DNA in eucaryotes 2, 3, In general, however, on kinetic and other grounds it is advantageous to use 5 short DNA fragments less than 500 base pairs in lengthS. This length not only permits separation of the repetitive and nonrepetitive components of most eucaryotic genomesl, 2 6, but also reduces the amount of single strand tails in reassociated and hybrid duplexes 7, 8, 9 A variety of methods have been employed to shear high molecular weight to DNA fragments of a particular desired size. One of the most common methods is to force a DNA solution through the valve of a high pressure cell 5 at a constant pressure of between 30,000 and 50,000 lbs/in 2 . DNA fragmented in this manner is typically reported to be 450 base pairs in length .
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2985
Nucleic Acids Research as determined from measurement of sedimentation velocityl 2, 3, 5 9. The results presented here show that DNA sheared at 30,000 lbs/in2 is considerably smaller than the 450 base pairs typically reported. DNA sheared in this manner has a mean fragment length of 220-235 base pairs as determined by gel electrophoresis and adsorption grid electron microscopy. Comparison of 5 sizing techniques indicates that both velocity sedimentation and Kleinschmidt electron microscopy overestimate mean fragment size in sheared DNA preparations. The implications of these observations are discussed as they relate to the current reassociation and hybridization technology. METHODS AND MATERIALS Preparation of DNA
Nuclei were prepared by homogenizing chick embryonic brain tissue in 0.075 M NaCl, 0.025 M EDTA, pH 8 (Buffer A) at 40C with 1% NP-40 (Shell) in a loose fitting dounce homogenizer. Cell breakage was virtually complete and nuclei were pelleted at 3,000 RPM for 10 min (Sorvall HB-4) and resuspended in Buffer A without NP-40 using a dounce homogenizer. The nuclei were again pelleted and resuspended in 0.1 M NaCl, 0.05 M Tris pH 7.4, 0.001 M EDTA (Buffer B); SDS was added to a final concentration of 1% and the suspension homogenized. An equal volume of phenol saturated in Buffer B was added and the mixture shaken at room temperature for 20 minutes and then centrifuged at 5,000 RPM for 10 min at 20°C. The aqueous phase was removed and extracted twice more with phenol as above and then extracted once with chloroform: octanol (8:1) to remove phenol and precipitated overnight at -20°C with 2.5 volumes of ethanol. The DNA precipitate was pelleted at 10,000 RPM for 30 min at -10°C and redissolved in Buffer B at a concentration of
Nucleic Acids Research
Sheared DNA fragment sizing: comparison of techniques
Charles P. Ordahl, Thomas R. Johnson and Arnold I. Caplan Departments of Biology and Anatomy, Case Western Reserve University, Cleveland, OH 44106, USA
Received 2 August 1976 ABSTRACT DNA fragmented by conventional French press shearing procedures (30,000 lbs/in2) has a number-average fragment size of 230 base pairs. This is considerably smaller than the 450 base pairs typically reported for DNA sheared by this method. Comparison of 5 sizing techniques indicates that sheared DNA fragment size is overestimated by either measurement of velocity sedimentation or Kleinschmidt Electron Microscopic visualization. Both adsorption grid electron microscopic visualization and gel electrophoresis yield the most reliable estimates of the mean size of small DNA fragment populations. In addition, the assessment of fragment size distribution (not possible from sedimentation analysis) potentially allows more critical evaluation of DNA hybridization and reassociation kinetic and measurement parameters. INTRODUCTION DNA reassociation and hybridization analysis requires the use of fragmented DNA Precise information regarding the size of the DNA fragments is essential to interpretation of these kinds of experiments. Fragment size is not only an integral element in the kinetics of DNA reassociation and hybridization, but also affects the kinds of DNA-DNA or DNA-RNA duplexes obtained. For some experiments it is necessary to use long (>1000 base pair) fragments of DNA. Fragments of this size have been used to determine the extent of inter4 spersion of repeated and nonrepeated sequences of DNA in eucaryotes 2, 3, In general, however, on kinetic and other grounds it is advantageous to use 5 short DNA fragments less than 500 base pairs in lengthS. This length not only permits separation of the repetitive and nonrepetitive components of most eucaryotic genomesl, 2 6, but also reduces the amount of single strand tails in reassociated and hybrid duplexes 7, 8, 9 A variety of methods have been employed to shear high molecular weight to DNA fragments of a particular desired size. One of the most common methods is to force a DNA solution through the valve of a high pressure cell 5 at a constant pressure of between 30,000 and 50,000 lbs/in 2 . DNA fragmented in this manner is typically reported to be 450 base pairs in length .
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2985
Nucleic Acids Research as determined from measurement of sedimentation velocityl 2, 3, 5 9. The results presented here show that DNA sheared at 30,000 lbs/in2 is considerably smaller than the 450 base pairs typically reported. DNA sheared in this manner has a mean fragment length of 220-235 base pairs as determined by gel electrophoresis and adsorption grid electron microscopy. Comparison of 5 sizing techniques indicates that both velocity sedimentation and Kleinschmidt electron microscopy overestimate mean fragment size in sheared DNA preparations. The implications of these observations are discussed as they relate to the current reassociation and hybridization technology. METHODS AND MATERIALS Preparation of DNA
Nuclei were prepared by homogenizing chick embryonic brain tissue in 0.075 M NaCl, 0.025 M EDTA, pH 8 (Buffer A) at 40C with 1% NP-40 (Shell) in a loose fitting dounce homogenizer. Cell breakage was virtually complete and nuclei were pelleted at 3,000 RPM for 10 min (Sorvall HB-4) and resuspended in Buffer A without NP-40 using a dounce homogenizer. The nuclei were again pelleted and resuspended in 0.1 M NaCl, 0.05 M Tris pH 7.4, 0.001 M EDTA (Buffer B); SDS was added to a final concentration of 1% and the suspension homogenized. An equal volume of phenol saturated in Buffer B was added and the mixture shaken at room temperature for 20 minutes and then centrifuged at 5,000 RPM for 10 min at 20°C. The aqueous phase was removed and extracted twice more with phenol as above and then extracted once with chloroform: octanol (8:1) to remove phenol and precipitated overnight at -20°C with 2.5 volumes of ethanol. The DNA precipitate was pelleted at 10,000 RPM for 30 min at -10°C and redissolved in Buffer B at a concentration of
