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

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

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

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

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)

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

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

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

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

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

Gel electrophoresis

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

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

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

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

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 2 3 1 23 Concepts of Genetics Klug & Cummings, Pearson Education

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

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

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

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)

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

* RFLP: detection of DNA polymorphism normal mutant Globin probe

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

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

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

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

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

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.

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

Dot blot hybridisation Microscopic slide automatic ‘printer’  micro-array: e.g.10.000 probes/cm2

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)

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

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)