Transcript Ch6AFLPRAPDSTR

Chapter 5: Hybridisation & applications
Hybridisation
Southern
In situ
RFLP
micro-array & SNP analysis
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Hybridisation
•The double-stranded DNA
molecule is a very stable
structure
•Strands can be separated by
high temperature, high pH (in
vitro) or enzymes (in vivo)
•When temperature is lowered
again, complementary strands
will automatically find each
other and hybridise , even is a
very complex mixture of
thousands of DNA fragments
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Hybridisation
• Hybridisation: process in which a labeled
single stranded piece of DNA (RNA) finds
(and basepairs with) the complementary
strand
• Probe: piece of
single-stranded DNA
(or RNA) that is labeled (
)
to detect the corresponding
complementary sequence in hybridisation 3
Hybridisation
• DNA denaturation = strand separation
occurs by heat to break hydrogen bonds
between DNA bases
• DNA renaturation =
hybridization =
complementary single
strands pair and
hydrogen bonds form
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Hybridisation
• The annealing (= hybridisation) temperature of
the DNA strands depends on the length, the
GC-content and the buffer conditions
• The length only matters for small molecules
(like primer annealing in PCR)
• Choosing different temperatures for the same
probe in the same buffer results in stringent
(higher temperature) or non-stringent
hybridisation (lower temperature)
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Hybridisation
Hybridisation
conditions can
be choosen to
have
• Perfect match
• Allow
mismatch
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Hybridisation
Hybridisation can be done in liquid
condition, but it is usually done with a
target DNA on a membrane and the
probe in solution (or reverse)
Examples:
• Colony hybridisation (petridish 
membrane
• Southern (gel  membrane)
• Dot blot, array, chip (membrane,
glass slide, chip)
• In situ hybridisation 1. WISH (whole
mount ISH) on tissue or
2. on chromosome spreads
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Hybridisation
Probe can be made on the basis
of:
• amino acid sequence of the
protein
• sequence known from another
organism
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Restriction enzymes
Restriction
enzymes
cut DNA
into defined
pieces,
named
restriction
fragments
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Gel electrophoresis
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Hybridisation
Southern (or DNA blot)
Too many bands on a gel???
Blot and hybridisation
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Hybridisation
Southern (or DNA blot)
• DNA bands on a gel can often be visualized
by staining with dyes which bind DNA
(ethidium bromide)
• Southern blot analysis is used to detect very
small amounts of DNA or to identify a single
DNA band in a complex mixture
• Southern blots use
labeled “probes” to
identify bands by
hybridization to
complementary
DNA bases
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Hybridisation
Southern (or DNA blot)
Steps in Southern blot procedure:
• DNA is cut into pieces by restriction
enzymes
• DNA fragments are separated by gel
electrophoresis
• DNA is denatured (by alkali) to produce
single-strand bands of DNA and
transferred from gel to hybridization filter
=blot procedure
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Hybridisation
Southern (or DNA blot)
• Filter is mixed with radiolabeled (or
otherwise labeled) single-stranded DNA
probe complementary to the DNA
sequence at temperatures which permit
hybridization = hydrogen bonds form
between complementary base pairs
• DNA bands hybridized to probe are
detected by X-ray film exposure
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Hybridisation
Southern (or DNA blot)
• Simple example: restriction digest with 3 fragments
• 3 different blots each hybridised with one of the
fragments
• Only the fragment that corresponds to the probe is
Probe 1
2
3
visible on that blot
1
2
3
Concepts of Genetics Klug & Cummings,
Pearson Education
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Hybridisation
• Instead of using a membrane with DNA or
RNA as a target, hybridisation can also be
done on cells and tissues = in situ
hybridisation
• On chromosomes (DNA)
• To detect RNA in tissues (expression
analysis)
Radioactive in situ
on nematode
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Detection of hybridised probe
• Radio-activity  autoradiography
• Fluorescence
• Enzymatic reaction with production of
coloured precipitate vb. BCIP/NBT
Fluorescent in situ on nucleus
In situ on animal tissue section
with colour precipitate
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RFLP: application of southern analysis
Restriction fragment length polymorphism:
Difference in length of restriction fragments
caused by differences in DNA sequence
between two alleles (of one individual or
between individuals).
Example:
-globin
alleles
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RFLP: detection of DNA polymorphism
DNA polymorphism
= difference in DNA
sequence
= due to mutation
Example: sickle cell
anemia: A T
mutation in globin
(oxygen carrying
protein)
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RFLP: detection of DNA polymorphism
This mutation causes a painful and deadly disease,
frequent in certain parts of Africa because it protects
against malaria, also in heterozygous
condition
Sicklecell anemia:
1 AA  in -globin
due to one base
mutation
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RFLP: detection of DNA polymorphism
Globin probe
normal
mutant
DNA
polymorphism
*
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RFLP: detection of DNA polymorphism
1. Isolate DNA (from
blood)
2. Restriction digest
3. Blot
4. Probe with globin
5. Analyse bands
DNA
polymorphism
DNA Science, eds. Midlos & Freyer, CSH Lab. Press
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Detection of DNA polymorphism
• Restriction fragment length polymorphism is
one of the (older) methods to detect DNA
polymorphisms (DNA marker). This method is
based on restriction and hybridisation,  it is time
consuming to develop and to perform on samples,
needs a lot of DNA, other methods are based on
PCR so much less DNA needed (chapter 6).
• Another newer hybridisation method is using
allel specific oligonucleotide probes (ASO) to
detect single nucleotide polymorphisms (SNP).
This is usually done on dot blot or chip
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Dot blot hybridisation with ASO
• DNA (for example genomic DNA of
humans or plants) is put on a membrane
as drops, denatured and fixed
• ASO = allel-specific oligonucleotide ca.
20 nt long
• Stringent hybridisation can distinguish
alleles that differ in only one nucleotide
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Dot blot hybridisation with ASO
DNA sequence of a heterozygote
Dot blot hybridisation with 2 ASO’s
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Dot blot hybridisation
• Dot blots exist in
different densities
(depending on drop
sizes)
• Low density dot blot
on nylon membrane:
manual or automatic
‘spotter’
 Macro-array eg.
600 spots/ 100 cm2
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Dot blot hybridisation
Dot blots exist in different varieties depending on the
probe types:
• Probes in solution, probes are labeled, only one
probe can be tested per hybridisation
 many targets (e.g. genomic DNAs of individuals)
possible on one membrane.
For examples see previous slides.
• Probes on the membrane, probes are not labeled,
labeled genomic DNA is added in solution (only one
individual is tested per hybridisation)
 many probes or SNPs can be tested on one
membrane = Reverse dot blot
For examples see further slides: micro-array and chips.
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Dot blot hybridisation
One probe in solution
 many targets (e.g.
genomic DNAs of
individuals) possible
on one membrane.
probe e.g. ASO
Labeled genomic DNA in solution
Probes on the membrane  many
probes or SNPs can be tested on
one membrane = Reverse dot blot
Labeled genomic DNA
DNA1 DNA2 DNA3
probe1 probe2
probe3
DNA4 DNA5 DNA6
probe4 probe5 probe6
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Dot blot hybridisation
Microscopic slide
automatic ‘printer’
 micro-array:
e.g.10.000 probes/cm2
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DNA chip hybridisation
www.affymetrix.com/technology
• Affymetrix chip: in situ synthesis of the
oligonucleotide probes: 106/chip (1,28cm)2
iGenetics 2001, Peter Russell
(Benjamin Cummings) 31
DNA chip hybridisation
Micro-array or chip for SNP analysis
Many thousand oligonucleotides are put on a slide
or chip, each time different alleles per locus. In one
hybridisation the genotype of an individual (labeled
genomic DNA in solution) can be analysed.
Hybridisation is done with
fluorescently labeled probe and
analysis via microscope, with a
computer for image processing
 enormous amount of information
in single experiment (whole genome)
 development of micro-arrays
enormous work and its use is
expensive
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SNP
Some methods for SNP- analysis
• RFLP
• Sequence-analysis (mini-sequencing e.g. via Mass spec)
• ASO-hybridisation via
- Dot blot
- Chip
• Use ASO as primer for PCR with stringent
annealing temperature (only PCR product when
perfect match of primer)
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