(9.2.1)--12-2MolecularBiologyTechniquesCo.pdf

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1、Chapter 12 Gene Diagnosis 12.2 Molecular Biology Techniques Commonly Used in Gene Diagnosis(Nucleic Acid MolecularHybridization and Biochip)12.2.1 Process of Nucleic Acid MolecularHybridizationTested Nucleic Acids PreparationBlotting and Fixation on a Hybridization MembraneHybridizationRemove Unhybr

2、idized ProbesDetection of Hybridization SignalsProbe PreparationProbe LabelingAdd Labeled ProbeThe DNA to be tested is prepared successively byrestriction endonuclease digestion,electrophoresisseparation,denaturation,and blotting.Afterhybridization with the probe,the presence of the DNAto be tested

3、is analyzed.12.2.2 Southern BlottingThis method can be used to detect geneticmutation and polymorphisms of small satellite oricrosatellite genetic markers.It has been widely usedin geneticdiseases diagnosis and forensicidentification.12.2.2 Southern BlottingDNA Fingerprint Analysis in Forensic Ident

4、ificationAfter electrophoresis seperation through adenaturring agarose gel,RNAs are bloted to amembrane and detected by hybridization.It can beused for Quantitative and qualitative analyses ofmRNAs or non-coding RNAs.12.2.3 Northern Blotting12.2.3 Dot Blot HybridizationRNAs or DNAs are denatured and

5、 directly loadedon the hybridization membrane for the qualitativeand quantitative study of genes and theirexpressed products.Sample Loading and Results of Dot Blot Hybridization 12.2.4 Reverse Dot BlottingThe unlabeled probes are first fixed on thehybridization membrane,and then hybridized withlabel

6、ed nucleic acid samples.Reverse dot blotblotting can simultaneously screen out multiplemutations in the samplesMembrane Having ProbesLabeled SampleReverse Dot Blotting Diagram Allele specific oligonucleotide(ASO)probes canbe used to significantly improve the reliability ofpoint mutation detection re

7、sults during(Reverse)dot hybridization.Because base mismatches inoligonucleotides can affect the stability of hybridduplex,apairofASOprobesareartificiallysynthesized.12.2.5 Allele-specific oligonucleotide probesN,Normal homozygote H,HeterozygoteM,Mutant homozygoteApplications of ASO probesNHMASO-NAS

8、O-MNormalMutantIncomplete HybridizationIncomplete HybridizationResults of Dot Blotting using ASO probesDNA to be testedNormalHaving Two Mutations12ASO-N12ASO-M12121212Applications of ASO probesResults of Reverse Dot Blotting using ASO probesABCD12.2.6 In Situ HybridizationIn situ hybridization is pe

9、rformed directly in fixedcells or tissues.It is used to detect the distributionand the copy number changes of target nucleic acidsin cells or tissues.It has been widely used in clinicalprenatal diagnosis.Representaitve Results of In Situ Hybridization in Clinical Prenatal Diagnosis12.2.7 BiochipGene ChipProtein ChipBiochips are used to detect DNAs,RNAs and proteins in high throughput.Thank you!