Pulse field gel electrophoresis (2)
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PULSED FIELD GEL ELECTROPHORESIS(PFGE) COURSE TEACHER PRESENTED BY DR. N. ERANNA MONOJ SUTRADHAR ASST PROF JR. M.SC,PANT BIOTECH PALB 3243 Dept of Plant Biotechnology University of Agricultural Science(UAS),Bangalore
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Transcript of Pulse field gel electrophoresis (2)
PULSED FIELD GEL ELECTROPHORESIS(PFGE)
COURSE TEACHER PRESENTED BY
DR. N. ERANNA MONOJ SUTRADHAR
ASST PROF JR. M.SC,PANT BIOTECH
PALB 3243Dept of Plant Biotechnology University of Agricultural Science(UAS),Bangalore
PULSE FIELD GEL ELECTROPHORESIS• Pulsed field gel electrophoresis is a technique
used for the separation of large deoxyribonucleic acid (DNA) , RNA, or protein molecules by applying to a gel matrix an electric field that periodically changes direction.
CONTI………..o Conventional gel electrophoresis of DNA
molecules is carried out by placing DNA in a solid matrix (i.e. agarose or polyacrylamide) and inducing the molecules to migrate through the gel under a static electric field.
o DNA fragments from 100 to 200 bp up to 50 kilobase pairs (kb) are separated by conventional gel electrophoresis techniques.
LIMITATIONS
• The gels used are extremely fragile due to the very low agarose concentrations, and the separation is not adequate for most applications.
• DNA(>50kb) cant be separated by this method.
PFGE-INTRODUCTION In 1982, Schwartz introduced the concept that DNA
molecules larger than 50 kb can be separated by using two alternating electric fields.
PFGE separates DNAs from a few kb
to over 10 Mb pairs
In conventional gels, the current is applied in a single direction (from top to bottom).
But in PFGE, the direction of the current is altered at a regular interval.
RELATED TERMS Pulsed Field - any electrophoresis process that uses more than one
electric field alternating
Switch Interval - amount of time by which each of the alternating fields is active
Reorientation Angle - acute angle between the two alternating electric fields
Field Inversion - PFGE system in shich the two alternating fields are oriented opposite each other
Voltage Gradient - electrical potential applied to the gel
Homogeneous Field - electric field that has uniform potential differences across the whole field
DESIGNS1)Orthogonal-Field Alternation Gel
Electrophoresis (OFAGE)
2)Transverse-Alternating Field Gel Electrophoresis (TAFE):
3)Field inversion gel electrophoresis(FIGE)
4)Rotating Gel Electrophoresis (RGE)
5)Contour-Clamped Homogeneous Electric Fields (CHEF)
PARTS OF PFGE SYSTEM1)Gel box
2)High voltage power supply
3)Switch unit
4)Computer system
RUNNING CONDITIONS FOR PFGEPulse Time. In PFGE, DNA is subjected alternately to two electrical
fields at different angles for a time called the pulse time.
Different DNA molecules have different pulse time
Electrical Field Strength
Electrophoretic mobility is defined as the velocity per unit field.
In most ordinary electrophoresis, the mobility is independent of field strength.
CONTIII
Temperature: In conventional gel electrophoresis, DNA molecules were run at room temperature.(PFGE-4oC-15OC)
Switch interval The highest resolution for molecules of a given size is obtained by using the shortest switch intervals
Agarose Concentration: Faster DNA migration occurs in gels of lower agarose concentration
APPLICATIONS OF PFGE
PFGE has proved to be an efficient method for genome size estimation
In PFGE DNA fragments obtained by using endonucleases produce a discrete pattern of bands useful for the fingerprinting and physical mapping of the chromosome.
The PFGE technique is useful to establish the degree of relatedness among different
strains of the same species.
PFGE has proven extremely powerful in the analysis of large DNA molecules from a variety of sources including intact chromosomal DNAs from fungi (16), parasitic protozoa.
Yeast Artificial Chromosome (YAC) libraries have been constructed by PFGE.
PFGE has also shown itself useful in the study of radiation-induced DNA damage and repair, size organization
