1. Chapter 5: Hybridisation & applications
Southern
In situ
RFLP
micro-array & SNP analysis

2. 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

3. 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

4. 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

5. 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)

6. Hybridisation Hybridisation conditions can be choosen to have
Perfect match
Allow mismatch

7. 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 on chromosome spreads

8. Recombinant DNA: Screening
Hybridisation
Recombinant DNA: Screening
Colony hybridisation is used to identify bacterial colonies containing the gene of interest
Colonies are
transferred to
filter, lysed,
DNA is denatured
by high pH
Membrane is then
hybridised with
labeled DNA from
gene to be cloned

9. Hybridisation Probe can be made on the basis of:
amino acid sequence of the protein
sequence known from another organism

10. Restriction enzymes
Restriction enzymes cut DNA into defined pieces, named restriction fragments

11. Gel electrophoresis

12. Southern (or DNA blot) Hybridisation
Too many bands on a gel??? Blot and hybridisation

13. Southern (or DNA blot) Hybridisation
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

14. Southern (or DNA blot) Hybridisation 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

15. Southern (or DNA blot) Hybridisation
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

16. Southern (or DNA blot) Hybridisation
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 visible on that blot
Probe 1 23
Concepts of Genetics Klug & Cummings, Pearson Education

17. 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

18. 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

19. 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

20. 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)

21. Sicklecell anemia: 1 AA  in -globin due to one base mutation
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

22. * RFLP: detection of DNA polymorphism normal mutant Globin probe

23. RFLP: detection of DNA polymorphism
Isolate DNA (from blood)
Restriction digest
Blot
Probe with globin
Analyse bands
DNA polymorphism
DNA Science, eds. Midlos & Freyer, CSH Lab. Press

24. 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

25. 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

26. Dot blot hybridisation with ASO
DNA sequence of a heterozygote Dot blot hybridisation with 2 ASO’s

27. 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

28. 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.

29. Dot blot hybridisation
One probe in solution
 many targets (e.g. genomic DNAs of individuals) possible on one membrane.
Labeled genomic DNA in solution
Probes on the membrane  many probes or SNPs can be tested on one membrane = Reverse dot blot
probe e.g. ASO
Labeled genomic DNA
DNA1 DNA2 DNA3
DNA4 DNA5 DNA6
probe1 probe2 probe3
probe4 probe5 probe6

30. Dot blot hybridisation
Microscopic slide automatic ‘printer’
 micro-array: e.g probes/cm2

31. DNA chip hybridisation
Affymetrix chip: in situ synthesis of the oligonucleotide probes: 106/chip (1,28cm)2
iGenetics 2001, Peter Russell (Benjamin Cummings)

32. 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

33. 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)