用荧光原位杂交技术构建高分辨率的DNA物理图谱(2)

核糖体(ribosomal)DNA的组织结构,能够直接排列和定位1kb左右,检测信号的灵敏度低于700bp,所得到的DNA纤维的分子结构都十分类似,DNA)都在3.0～3.5kb/μm之间(Wiegant等,1992;Houseal等,1994;;1995;Fransz等,1996)。这一数值已十分接近Watson和Crick建立的

94kb/μm,这说明所得到的DNA纤维很可能已是分离出的没有蛋白质复合的DNA综上所述,由于所使用的靶DNA切片的染色体或DNA分子结构的不同,FISH技术所能达到的分辨率水平有很大的差别。以番茄染色体为例,粗线期染色体的浓缩度比中期染色体要低大约10-15倍,而DNA纤维比中期染色体低900倍左右。这里我们对各种FISH技术的特点,包括分辨率水平、可检测DNA序列的范围、以及优点和缺点进行了总结和比较(见表1)。

高分辨率的DNA荧光原位杂交技术能够快速准确地得到探针序列之间的顺序、方向和真实的物理距离。在具体应用这一技术时,根据不同染色体和DNA纤维的结构特征,选择适当的靶DNA载体是很重要的。根据我们的研究结果认为,如果从性细胞中制备粗线期染色体在技术上可行的话,在这种染色体切片上进行FISH,可以直接准确地找出与目的基因相关的DNA探针克隆在染色体特定区域内的定位,确定相距100kb以上的DNA序列之间的顺序和相互位置关系。一旦目的基因的范围被缩小到100kb以後,可以直接在DNA纤维上进行FISH,快速准确地构建出这一区域的DNA物理图谱。把在粗线期染色体和DNA纤维上的FISH技术与现有的分子生物学技术相结合,将有效地加速大范围DNA物理图谱的构建,以及分离和克隆目的基因的进程。

参　考　文　献

1　DauwerseJG,WiegantJ,etal.Multiplecolorsbyfluorescenceiinsituhybridizationusingratio-labelledDNAprobescreateamolecularkaryotype.HumanMolecularGenetics,1992,1:593～598

2　FlorijnRJ,BondenLAJ,etal.High-resolutionDNAfiber-FISHforgenomicDNAmappingandcolourbar-codingoflargegenes.HumanMolecularGenetics,1995,4:831～836

3　FranszPF,Alonso-BlancoC,etal.High-resolutionphysicalmappinginArabidopsisthalianabyfluorescenceinsituhybridizationtoextendedDNAfibres.ThePlantJournal,1996,9:～430

4　GanalMW,LapitanNLV,TanksleySD.Macrostructureofthe1991,3:87～94

5　HaafT,WardD.StructuralanalysisofasatelliteDNAchromatinandchromosomes.HumanMolecularGenetics,1994,3:697～709

6　HeiskanenM,KarhuR,etal.iinsituhybridizationtoextendedDNAfiberspreparedfromagarose-,,17:928～933

7　HeiskanenM,andA.VisualmappingbyhighresolutionFISH.TrendinGenetics,1996,12:379～3828　HengHHQ,,A,1992,89:9509～9513

9　HousealTW,DackowskiWR,etal.Highresolutionmappingofoverlappingcosmidsbyfluorescenceiinsituhybridization.Cytom2etry,1994,15:193～198.

10　InazawaJ,AriyamaT,etal.HighresolutionorderingofDNAmarkersbymulti-colorfluorescenceiinsituhybridizationof

prophasechromosomes.CytogeneticsandCellGenetics,1994,65:130～135

11　JiangJM,GillBS.Nonisotipiciinsituhybridizationandplantgenomemapping:thefirst10years.Genome,1994,37:717～

725

12　JoosS,FinkTM,etal.Mappingandchromosomeanalysis:thepotentialoffluorescenceiinsituhybridization.JournalofBiotech2

nology,1994,35:135～153

13　LawrenceJB,VillnaveCA,SingerRH.Sensitivehigh-resolutionchromatinandchromosomemappingiinsitu:presenceand

orientationoftwocloselyintegratedcopiesofEBVinalymphomaline.Cell,1988,52:51～61

14　LawrenceJB,SingerRH,McNellJA.Interphaseandmetaphaseresolutionofdifferentdistanceswithinthehumandystrophin

gene.Science,1990,249:928～932

15　LichterP,TangCJC,etal.High-resolutionmappingofhumanchromosome11byiinsituhybridizationwithcosmidclones.

Science,1990,247:64～69

16　ParraIandWindleB.HighresolutionvisualmappingofstretchedDNAbyfluorescencehybridization.NatureGenetics,1993,5:

17～21

17　RamannaMSandPrakkenR.Structureofandhomologybetweenpachyteneandsomaticmetaphasechromosomesofthetomato.

Genetica,1967,38:115～133

18　RiedT,BaldiniA,etal.SimultaneousvisualizationofsevendifferentDNAprobesbyinsituhybridizationusingcombinatorialfluo2

A,1992,89:1388～1392

19　ShermanJDandStackSM.Physicalmapofcrossoverfrequencyonsynaptonemalcomplexesfromtomatoprimarymicrosporocytes.

TGCreport,1995,45:42～43

20　TraskB,PinkelD,vandenEnghG.TheproximityofDNAsequencesininterphasecellnucleicorrelatedtogenomicdistanceand

permitsorderingofcosmidsspanning250kilobasepairs.Genomics,1989,5:710～717

21　TraskB,MassaHF,BurmeisterM.Fluorescenceinsituhybridizationestablishestheordercen-DXS28(C7)-DXS67(B24)-

DXS68(L1)-telinhumanchromosomeXp21.3.Genomics,1992,13:455～457

22　WiegantJ,KalleW,MullendersL,etal.High-resolutioniinsituhybridizationusingDNAhalopreparations.HumanMolecular

Genetics,1992,1:587～591

23　ZhongXB,deJongJH,ZabelP.Preparationoftomatomeioticpachyteneandmitoticmetaphasechromosomessuitableforfluores2

cenceiinsituhybridization(FISH).ChromosomeResearch,1996a,4:24～28

24　ZhongXB,FranszPF,etal.HighresolutionmappingonpachytenechromosomesandextendedDNAfibresbyfluorescenceiin

situhybridization.PlantMolecularBiologyReporter,1996b,14(3):232～242

1996-11-04收稿,1997-01-23修回.