Using molecular marker technology in studies on plant genetic diversity Complementary technologies
Copyright: IPGRI and Cornell University, 2003
Contents
! Denaturing gradient gel electrophoresis (DGGE)! Thermal gradient gel electrophoresis (TGGE)! Single-stranded conformational polymorphism
! Heteroduplex analysis! Denaturing high-performance liquid
Copyright: IPGRI and Cornell University, 2003
Introduction
! Various technologies serve to show the presence
! They help narrow down the possibilities of what
we need to sequence (is there apolymorphism that might be worth sequencing?)
! But they do not identify what the sequence
Copyright: IPGRI and Cornell University, 2003
When looking at differences in the DNA sequence, we need to be able to separatespecific DNA segments from a mixture such as from the whole genome.
Electrophoresis separates molecules in an electrical field on the basis of charge, sizeand shape. If a DNA molecule is cut into small sections and placed in a well at one end(cathode) of an agarose gel, the DNA fragments will move through the gel towards theanode. Their speed will depend on their individual sizes, so they end up forming bandslocated at different positions in the gel. The bands can then be visualised with ethidiumbromide staining, which causes the DNA to fluoresce under UV light.
The same result is achieved by electrophoresis in an acrylamide gel, the differencebeing a matter of resolution. The acrylamide is able to discriminate smaller differences infragment size.
Electrophoresis is a diagnostic procedure that allows us to identify molecules of differentsizes. When used as such, electrophoresis is itself a means of showing polymorphismsand, consequently, genetic variation between genotypes.
But electrophoresis can also be useful as a first step towards identifying and isolatingspecific DNA molecules that, even if the same size, differ in sequence composition. Denaturing gradient gel electrophoresis (DGGE) (1)
! Permits detecting very small DNA polymorphisms
! Does not require previous knowledge on the
! Based on the renaturation properties of DNA
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Denaturing gradient gel electrophoresis (2)
electrophoresis through a polyacrylamide gelunder increasingly denaturing conditions(formamide/urea concentrations)
! The DNA 'melts' and becomes single stranded
! DNA molecules change their shape and stop
! Differences in DNA sequence are identified
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The DNA fragments migrate first as double-stranded molecules. Later, because of thegel's changing composition, the molecules denature and become single stranded,forming a branched structure. This changed structure results in the molecules'diminished ability to move through the gel.
The point at which the DNA melts depends on the nucleotide sequence in the meltedregion. The final location of the molecules in the gel thus depends on the nucleotidesequence of the fragments. Denaturing gradient gel electrophoresis (3)
conditions by the ‘melting point’ process
When samples are mixed, double bands indicatesequence differences between bands of thesame size
Copyright: IPGRI and Cornell University, 2003
At least 95% of differences in sequence composition is estimated as being detectedwith this procedure.
DGGE also serves to distinguish homozygous versus heterozygous genotypes for aparticular DNA fragment. To take advantage of this capacity, a cycle of denaturation andrenaturation must be conducted after the last PCR cycle. Homoduplexes andheteroduplexes are formed as alleles reassociate. In the DGGE gel, fast-migratinghomoduplex combinations will indicate homozygous genotypes. Heterozygousgenotypes will show both homoduplex and heteroduplex combinations. Heteroduplexesare formed through mispairing and rapid denaturation in the gel, which will stop themigratory course of these molecules. Thermal gradient gel electrophoresis (TGGE)
! Increasingly denaturing conditions are achieved
by a temperature gradient instead of bychanging reagent concentrations
! Can also be used for analysing single-stranded
Copyright: IPGRI and Cornell University, 2003
Basic reference
Myers, R.M., N. Lumelsky, L.S. Lerman and T. Maniatis. 1985. Detection of single base
substitutions in total genomic DNA. Nature 313:495-498. Single-stranded conformational polymorphism (SSCP) (1)
! SSCP is based on the electrophoretic behaviour
of a single-stranded DNA molecule through anon-denaturing acrylamide gel
• A single-stranded molecule has the property
of forming secondary structures throughinternal base pairings
• These secondary structures are sequence-
dependent and result in particular shapesfor each single-stranded molecule
! Differences in secondary structures cause the
DNA strands to migrate differentially on the gel
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SSCP can distinguish between two very similar DNA sequences only on the basis of theparticular shape of their single-stranded structures. In principle, then, even two alleles ofthe same gene can be discriminated.
! The sequence of interest goes through PCR
! Next, the PCR product is denatured at 94°C and
rapidly cooled down on ice. Single-strandedmolecules do not pair but form stable secondarystructures
! The reassociated fragments are then subject to
• In an homozygous case, two bands can be observed,
each corresponding to a slightly different secondarystructure
• In a heterozygous case, at least four bands can be
Copyright: IPGRI and Cornell University, 2003
SSCP is a simple technique, but has at least two major disadvantages:
The electrophoretic behaviour of the single-stranded molecules isunpredictable, depending very much on temperature and runningconditions.
In the case of long DNA fragments (> 200 bp), the method becomesinsensitive to some mutations. In principle, SSCP seems to work betterfor small insertions and/or deletions. Basic reference
Hayashi, K. 1992. A method for the detection of mutations. Genet. Anal. Tech. Appl. 9:73-79. Heteroduplex analysis
! Two PCR-amplified products are mixed in equal
quantities, denatured at 95°C and allowed tocool
! During cooling, DNA strands reanneal to form
! Any mismatches in the heteroduplex will cause it
to have a different three-dimensional structure,with a reduced mobility that is proportional to thedegree of divergence of the sequences
Copyright: IPGRI and Cornell University, 2003
Basic reference
Delwart, E.L., E.G. Shpaer, J. Louwagie, F. McCutchan, M. Grez, H. Rübsamen
Waigmann and J.I. Mullins. 1993. Genetic relationships determined by a heteroduplexmobility assay: analysis of HIV env genes. Science 262:1257-1261. Denaturing high-performance liquid chromatography (DHPLC): methodology
! This method can detect sequence differences of
a single base pair as well as insertions and/ordeletions
! Works with crude PCR products and does not
! Based on the differential elution of homoduplex
and heteroduplex DNA when run through acolumn
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DHPLC is a high-performance liquid chromatography method in which DNA fragments areseparated according to size and/or presence of heteroduplexes (reannealed DNAstrands) during their passage through a gradient in a column.
In double-stranded amplified DNA, nucleotides that are mismatched through mutationsand polymorphisms become evident after heteroduplex formation. The presence of thesepolymorphisms creates a mixed population of heteroduplexes and homoduplexes duringreannealing of wild type and mutant DNA. If this mixture of fragments is run underpartially denaturing conditions by HPLC, heteroduplexes elute from the column earlierthan the homoduplexes because of their lower melting temperature.
Analysis can be performed to detect sequence variation between individuals ordetermine heterozygosity. DHPLC: applications
! Finding new mutations and polymorphisms in
any DNA fragment or particular gene sequence
! Evaluating the fidelity of amplified fragments
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Basic reference
Oefner, P.J. and P.A. Underhill. 1998. DNA Mutation Detection Using Denaturing High-
Performance Liquid Chromatography (DHPLC). Current Protocols in Human Genetics,supplement 19, 7.10.1-7.10.12. Wiley & Sons, NY. In summary
! Several technologies help identify the presence
! While they cannot tell what the variant is, they
can help narrow the range of strategies to use fordetecting it
Copyright: IPGRI and Cornell University, 2003
By now you should know
! Denaturing gradient gel electrophoresis
! Single-stranded conformational polymorphism
Copyright: IPGRI and Cornell University, 2003
Final considerations
Copyright: IPGRI and Cornell University, 2003
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Using molecular marker technology in
Contents
Introduction
Denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis (2)
Denaturing gradient gel electrophoresis (3)
Thermal gradient gel electrophoresis (TGGE)
Single-stranded conformational
! The sequence of interest goes through PCR
! Next, the PCR product is denatured at 94°C and
rapidly cooled down on ice. Single-strandedmolecules do not pair but form stable secondarystructures
! The reassociated fragments are then subject to
• In an homozygous case, two bands can be observed,
each corresponding to a slightly different secondarystructure
• In a heterozygous case, at least four bands can be
Copyright: IPGRI and Cornell University, 2003
SSCP is a simple technique, but has at least two major disadvantages:
The electrophoretic behaviour of the single-stranded molecules isunpredictable, depending very much on temperature and runningconditions.
Heteroduplex analysis
Denaturing high-performance liquid
DHPLC: applications
In summary
By now you should know
Final considerations