CHAPTER SIX Nucleic acid hybridization: principles and applications 생물정보학협동과정 강민호

Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental tool in molecular genetics which takes advantage of the ability of individual single-stranded nucleic acid molecules to form double stranded molecules (that is, to hybridize to each other)

- A labeled nucleic acid - a probe - to identify related DNA or RNA molecules - Complex mixture of unlabeled nucleic acid molecules- the target -Base complementarity with a high degree of similarity between the probe and the target. Standard nucleic acid hybridization assays

Types of probes

Probes DNA labelling –5’ –3’ –Uniform labeling Nick translation Random primer PCR-mediated labeling RNA labelling –In vitro transcription of a cloned DNA insert Different probes –Radioactive labeling or isotopic labeling –Nonradioactive labeling or nonisotopic labeling

Kinase end-labeling of oligonucleotides

Fill-in end labeling

Nick translation

Random primed labeling

Riboprobes

Characteristics of radioisotopes commonly used for labeling DNA and RNA probes RadioisotopeHalf-lifeDecay-typeEnergy of emission 3 H12.4 years  MeV 32 P14.3 days  MeV 33 P25.5 days  MeV 35 S87.4 days  MeV

Nonisotopic labeling and detection The use of nonradioactive labels has several advantages: –safety –higher stability of a probe –efficiency of the labeling reaction –detection in situ –less time taken to detect signal Major types –Direct nonisotopic labeling ( ex. nt labeled with a fluorophore ) –Indirect nonisotopic labeling ( ex. biotin.-streptavidin system )

Structure of fluorophores Fluorescence microscopy Common Fluorophores

Structure of digoxigenin-modified and biotin-modified nucleotides

Indirect nonisotopic labeling

Nucleic acid hybridization- formation of heteroduplexes

Denaturation of DNA results in an increase of optical density

Factors affecting Tm of nucleic acid hybrids Destabilizing agents ( ex. formamide, urea ) Ionic strenght Base composition ( G/C%, repetitive DNA) Mismatched base pairs Duplex lenght Different equations for calculating Tm for: DNA-DNA hybrids DNA-RNA hybrids RNA-RNA hybrids Oligonucleotide probes

Stringency High temperatureLow salt concentrationHigh denaturant concentration High strigency Low strigency Low temperature Sequence G/C content Sequence lenght Tm Low denaturant concentration High salt concentration Perfect match complementary sequences Perfect match non-complementary sequences

The identification of specific sequences in a complex mixture.

Filter hybridization techniques Filter hybridization methods Bacteriophage blotting Benton-Davis Bacterial colony blotting Grunstein-Hogness Slot/Dot blotting Northern analysis Southern analysis

Filters or Membranes Nitrocellulose Nylon Positive charged nylon (hybond) PVDF ( hydrophobic polyvinylidene difloride ) Different properties: –Binding capacity (mg nucleic acids/cm 2 ) –Tensile strenght –Mode of nucleic acid attachment –Lower size limit for efficient nucleic acid retention

Dot blot or slot blot

Principles of Southern blot

Northern Blot

Colony blot hybridization

In situ hybridization Chromosome in situ hybridization –Metaphase or protometaphase chromosomes are probed with labeled DNA. The DNA can be labeled with a fluorochrome (FISH). Tissue in situ hybridization –Sliced or whole mounted preparations can be probed with RNA probes to detect mRNA expression

Tissue In situ hybridization

Gridded clone hybridization

Construction of DNA and oligo microarrays

Gene expression profiling by hybridization

Summary I Hybridization is due to complementarity of DNA strands. DNA can be labeled various ways Isotopic and non isotopic Hybridization can detect identical or similar sequences.

Summary II A variety of techniques utilize hybridization of DNA or RNA probes –ASO –Southern Blot, RFLP, VNTRs, Mutation detection, deletion detection –Northern Blot, tissue specific expression –In situ hybridization Chromosome location and integrity Tissue specific expression

Summary III Colony hybridization can be used to identify specific clones. Once you have one clone you can find others that hybridize to it. Screening of gridded clones. One can identify genomic clones homologous to a cDNA or identify cDNA expressed in a cell line. Microarrays can be used in many ways to analyze gene expression in various cell types, in response to various stimuli.