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They give us the opportunity to analyse vast amounts of genetic information in a single experiment. The use of this technique holds many possibilities, and developments are occurring daily. &     -The main applications of microarrays include:$.-((&     qComparative genome analysis Re-sequencing of DNA Functional genome analysis Developing knowledge of gene function"rq(( r  .Background to the Technique of DNA Microarrays"/.B"    f\ DThe success behind the technique of DNA microarrays can be attributed to the property that is responsible for the very existence of the DNA molecule. The DNA helix is held together by complementary base-pairing, and the exquisite specificity of this is responsible for, and exploited in, the technique of DNA microarrays. 0E@'       g]As can be seen in the above diagram, there are four different bases (A, G, C, T) and the hydrogen bonds between the complementary pairs of bases - shown below in blue - bind the DNA strand together.ES!   Hybridisation" $  Hybridisation is the basis behind the technique of DNA microarrays. The principle of the hybridisation reaction is to expose two single-stranded DNA sequences to each other, and measure the amount of double-strands that form. DC$&6    Microarray Fabrication"H     $Components of Microarray Fabrication %$&      The complete process involved in making DNA microarrays has the following main steps: preparation of samples construction of the arrays preparation of the probes hybridisation0VZVZ(&+   y Preparation of Samples$6  The first step in the process of microarrays is the preparation of the target DNA. This can be done from the genomic source, or Expressed Sequence Tags. 64$$J$&    o PI The Polymerase Chain Reaction is then used to amplify this DNA. This amplification step is vital in many applications and array formats. L(($(K$&     Construction of the Arrays$0  There are a number of methods for producing the microarray slides, and these are mainly done using robotic systems.They each have their own advantages and disadvantages. &/   q y` 8 The three main methods for constructing the arrays are:99$ 9 $ Microspotting Techniques"(    The use of prefabricated oligonucleotides greatly simplifies the construction of microarrays. Conventional methods can be used to produce the sequences, and these can then be printed directly onto the microscope slide (which is first overlaid with a coating that is positively charged). The cDNA or oligonucleotide material can be deposited straight from a reagent tray (where it is suspended in a denaturing solution) onto the glass surface by a printhead containing microspotting pins or micropipettes. "   '              ($Piezoelectric Printing$((  BMinute volumes of reagents are delivered to defined locations on the slide similar to  ink-jet printing methods. The printhead moves across the array, and at each spot electrical stimulation causes the DNA bases, cDNAs or other molecules to be delivered onto the surface via tiny jets. ""@x   V  E )%Photolithography(  *This makes use of semiconductor technologies. A mercury lamp is used, and the light activates modified photospecific versions of the four DNA bases. Photolithographic masks ensure the DNA synthesis is stimulated in defined positions - the masks predetermine which of the nucleotides are activated. This  in situ fabrication technique was developed by Affymetrix, and is used to produce their GeneChips.Pth     (       ,(Preparation of the Probes(66   Fluorescent probes must be prepared, and these hybridise to the microarray. These are prepared from messenger RNA from the cells or tissues of interest. Extraction of the mRNA is made easier by the identification of a key property by which the mRNA can be isolated.Z?   d  D   .* After the mRNA has been extracted from the cells or tissues under study, it is converted into DNA by the use of the reverse transcriptase enzyme. During this reaction, the DNA is labelled by the incorporation of fluorescent or radioactive nucleotides into the DNA. The two samples are labelled using two different fluorescent dyes - say, red or green. The most common dyes in use are Cy3 and Cy5.t"@   n    x_ This labelled DNA is then hybridised to the microarray slide. Using mixtures that are differentially labelled allows the ratio of fluorescence to be measured, therefore avoiding most of the problems of hybridisation kinetics. 0<$$&,    0, Hybridisation( 66  Hybridisation is the reaction that occurs between the fluorescent probes and the DNA on the microarray. The hybridisation conditions also depend on the application of the array: &[   N 1- After hybridisation, the microarray is scanned using a laser confocal scanning microscope, which illuminates each spot of DNA and separately measures the fluorescence for each dye. 67"]@     q 2.Microarray Equipment"H     3/Arrayers   Arrayers are available from a number of companies at a cost of approximately $60,000. The construction of arrayers is possible in the laboratory, but it is not easy to do. Some laboratories have relied on professional engineers. "4 c  t 60Scanners   NThe principle of scanners is to detect the different levels of fluorescence between the spots on the microarray. This produces a ratio of fluorescence, representing a ratio of hybridisation, for the different mRNA or cDNA sequences. This gives an indication of the expression of the genes under study in the tissue or cell samples. "OMZd   b    q 71 }A light source inside the scanner excites the fluorescently labelled samples. This is then detected, measured and recorded. ~~ &.   C :3Scanners commonly used include:& $   Molecular Dynamics Storm System Axon Instrument s GenePix 2000ZF&9   <4Data Analysis and Management"H   >6 After hybridisation and the readout of the expression levels obtained from the array, the vast quantities of data that is collected has to be stored and saved. This then enables processing and analysis - both statistical and biological - to be carried out on the data. This is vital, because without significant image processing, the information content of a microarray is very limited. &h    F?Oligonucleotide Arrays"H   G@ The DNA can be 'spotted' as intact pieces onto the membrane. An alternative method is one inspired by the computer chip industry - leading to the "DNA Chip" terminology. This technique is oligonucleotide arraying. $&    HA hOligonucleotide arrays are, in effect, miniaturised DNA microarrays and have evolved as an important tool in research into genomes. They are small, high-density arrays, containing tens of thousands of synthetic oligonucleotides. Oligonucleotide arrays differ in many details but do share the essential simplicity of the experimental design of DNA microarrays.ii (       f    JCUses of Oligonucleotide Arrays"&   Oligonucleotide arrays have a wide range of possible applications, and in a wide range of species. In many cases they have been employed for the same purposes as DNA microarrays. The main areas of application in which oligonucleotide arrays can speed up genomics studies are: 2  h    *     RLApplications of DNA Microarrays" H    SMIn Arabidopsis thaliana.&   DNA microarrays have been used to: measure differential expression between genes in wild-type and transgenic A. thaliana plants investigate gene expression in 1,443 genes, prepared from root and leaf mRNAh#" J UN   a   O  TN In Humans    DNA microarrays have been used to: study the gene expression characteristics of diseases identify new classes of cancer and to assign tumours to classes already described>##&    UOIn Yeast    DNA microarrays have been used to: identify functions of predicted Open Reading Frames in the genome of Saccharomyces cerevisiae study the effects of the diauxic shift - from anaerobic to aerobic metabolism - in S. cerevisiaep##FU h   Y    4   VPAdvantages of DNA Microarrays"H    WQ Being able to study the behaviour of many genes simultaneously is a great advantage. The technique is very fast: there can be as many as 150 copies of an array of 12,000 genes printed in only 1 day. DNA microarray technology is relatively cheap to use:`sW7sU6 &   ( XR 'A major advantage of DNA microarrays is that information about the sequence of the DNA is not required to construct and use the DNA microarrays. In fact, most of the human genes that have used microarray technology in expression studies are only defined by partial EST sequences at the moment."('Z   _   3   [ YSProblems of DNA Microarrays"H    ZTArray Fabrication   NWhen working on the kind of scale necessary for microarrays - micrometres - DNA can actually be very difficult to handle. It has been described as acting 'almost like concrete coated with superglue' (Marshall, 1998). To reduce these effects, a number of solutions are being developed: The electrical polarisation of the array spots, developed by Nanogen. The use of 'nonwettable' blank regions on arrays, created by microscopic surface tension barriers, by Protogene. However, simply using smaller chips, with the associated higher densities of sample, may prove to be the best option. v=i u /           N   y [U!Equipment and the Associated Cost""!( " fProviding the equipment necessary to carry out this technique remains a limiting factor. After the initial capital outlay, the cost in this technique comes from the lengthy experimental procedure. The need for so many cDNAs to be printed is also a disadvantage of this technique. To decrease the cost of this technique, the number printed can be reduced. gf&   \VLimitations of Equipment   ;As well as the cost of robotics to perform the technique, there may be technical limitations. The technique of DNA microarrays is currently limited not by the number of probes on an array, but by the resolution of the scanner used. This is helping to drive the development of more advanced systems and equipment."<; &t    ]WData Processing   6The development of methods that efficiently organise, distribute and interpret this data could be said to be the most formidable task for researchers. Currently in development is a software package known as LIMS - Laboratory Information Management Systems. This software and the databases associated with it: b7)5 7 ^XEthical Issues   Unfortunately, whilst having the potential for doing a great deal of good, the technique of DNA microarrays also raises a number of ethical problems. The DNA chip provides us with an extensive new range of applications for genetic diagnosis. The benefits of this include being able to determine the genetic factors of disease, but the drawbacks include more opportunities for genetic discrimination. &_   ' _YFuture Possibilities"H   a[ Fingerprints    TDNA microarrays will enable the identification of  fingerprints of gene expression. These fingerprints will categorise particular diseases, or other biological states. " &    w^DNA MICROARRAYS"H  'A third year project by Louise Asbury J(0#0'  "!0'(/\e{|}~      !"#$%&'()*+,-./0P/1b  ` ̙33` ` ff3333f` 333MMM` f` f` 3>?" dd@,|?" dd@   " @ ` n?" dd@   @@``PR    @ ` ` p>>'0 $(    6r P  T Click to edit Master title style! !  0t   RClick to edit Master text styles Second level Third level Fourth level Fifth level!     S  0y ``  X*  0L `   Z*  0 `   Z*H  0޽h ? ̙33 $Blank Presentation0 @(    0E P    T*    0J     V*  d  c $ ?    0F  @  RClick to edit Master text styles Second level Third level Fourth level Fifth level!    S   6P `P   T*    6V `   V*  H  0޽h ? ̙33  P4(  4 4 0` P    P*   4 0ܩ     R*   4 6 `P   P*   4 6 `   R*  H 4 0޽h ? ̙33'  :2 ( IT`T0   C 0 <$D 0  B  s *޽h ? ̙33p  :20@( w @ @ C d8 @ <$D 0  B @ s *޽h ? ̙338  N  @`( w   C <P<$ 0    0@3`@G Z3/50& 2    0?`4 f Introduction* 2   H  0޽h ? ̙33 PF  phP((  ( ( C hF<$D 0    ( C 4Ip<$  0   ( 0|J`@G Z4/50& 2   ( 0䰸`4 f Introduction* 2   H ( 0޽h ? ̙33p  @8`8( w 8 8 S   pd( Ow@   S X<$ 0    00Z`@G Z6/50& 2    0A`4 d Background* 2   H  0޽h ? ̙33 ]  :2(    S e 0 <$D  0     0A?0,$D  0  0A?P 0`,$D  0  0g`4 d Background* 2   H  0޽h ? ̙33 >A nfT( w T T C t<$D 0    T C t<$ 0   T 0u`@G Z8/50& 2   T 0z`4 d Background* 2   H T 0޽h ? ̙33 p  :2x( @Y\ x x C ` <$D 0  B x s *޽h ? ̙33  ]  ph|(  | | C T<$D 0    | C 4 <$D  0   | 0|`G [10/50& 2   | 0`4 e Fabrication* 2   H | 0޽h ? ̙33  6 #(    C P<$D 0     C <$D  0    0 `G [11/50& 2    0` 4  Fabrication - Sample Preparation8! 2 ! H  0޽h ? ̙33[  . t( w t t C x`<$D  0   t 0`G [12/50& 2   t 0D`0 4  Fabrication - Sample Preparation8! 2  ! H t 0޽h ? ̙33D  >A  l( w   C ȯ0<$D 0     C ` <$Dp 0  A  0$ `,$D 0 e The DNA sequences are printed onto the microscope slides robotically, in the specific grid pattern. ff f   0`G [13/50& 2    0`p4  Fabrication - Array Construction8! 2  ! H  0޽h ? ̙33) U 0Q( w 0 0 S  pp<$D 0   0 0x `,$D 0 ,Spotting of DNA fragments directly onto the slide Arraying of prefabricated oligonucleotides In-situ synthesis of oligonucleotides, done on the chipZL         0 0`G [14/50& 2   0 0`4  Fabrication - Array Construction8! 2  ! H 0 0޽h ? ̙33 ] #( w   C  P0<$D 0     C <$D 0    04`G [15/50& 2    0`4  Fabrication - Array Construction8! 2  ! H  0޽h ? ̙33 PF %( w   C $P<$D 0     C `` <$D 0    0\  ,$D 0 fThis is a non-contact process.    0`G [16/50& 2    0$`4  Fabrication - Array Construction8! 2  ! 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The hybridisation pattern can then be used to identify the genes that are expressed differently in the tissues or cells. $  H  0޽h ? ̙33p  :2(    C X <$D 0  B  s *޽h ? ̙33 ]   F( w   S dP<$D 0     S 0 <$D 0  ^  0  0,$D 0 ^It is more likely that these arrayers will become more affordable as the technology improves. "_]&  :   C rAZA:\microarrays\webpics\biorobmicrogridII.jpgp  ,$D 0  0ؙ`G [24/50& 2    0lP`4 c Equipment* 2   H  0޽h ? ̙33 `m nf(    C P<$D 0     C d0<$Dp 0    0`G [25/50& 2    00`4 c Equipment* 2   H  0޽h ? ̙33 PFA ~ ( s   S xP 0<$D  0   0 0A ?@ `; H $D 0   0L-,$D 0 fHow a scanner works... (    0T`G [26/50& 2    0`4 c Equipment* 2   H  0޽h ? ̙33& PF  N( s     S P<$D 0      S ` 0<$D  0    0 0A ?0 y H $D 0    c $A ?? p8 $D 0   0`G [27/50& 2     0T\`4 c Equipment* 2   H   0޽h ? ̙33p  @8( w   S  <$D 0  B  s *޽h ? ̙33:  N   b(      C 0<$D 0     0`G [29/50& 2     0d`4 g Data Analysis* 2   H   0޽h ? ̙33p  @8H( w H H S D <$D 0  B H s *޽h ? ̙33Q  N A Ly( JE, L L C @@<$D 0   L 0`G [31/50& 2   L 0|``4 ~Oligonucleotide Arrays* 2  H L 0޽h ? ̙33Q  U Py( w P P C <$D 0   P 0`G [32/50& 2   P 0``4 ~Oligonucleotide Arrays* 2  H P 0޽h ? ̙33  !e  XH( 87\ X X C P<$D 0    X C P <$D 0  ' X 0P  ,$D 0 KGenotyping Gene Expression Research into Diseases Mapping Genomic LibrariesLL L  X 0 `G [33/50& 2   X 0(``4 ~Oligonucleotide Arrays* 2  H X 0޽h ? ̙33"p  @80(    S 8 <$D 0  B  s *޽h ? ̙33 #U  -%@( w   S P<$D 0     S  <$D 0   0 0A ? 3H $D 0   0`G [35/50& 2    0$`4 f Applications* 2   H  0޽h ? ̙33 $PFA qiP( s   C 4P<$D 0     C 7<$D 0    09`G [36/50& 2    0̑`4 f Applications* 2   H  0޽h ? ̙33 %PF }u` (    S C <$D 0     S D0<$D 0    0E`G [37/50& 2    0|J`4 f Applications* 2   H  0޽h ? ̙33&p  @8p(    S XR <$D 0  B  s *޽h ? ̙33 '0u  z(    C W <$D 0    0X  ,$D  0 the initial cost of constructing an arrayer is approximately $60,000; after this, the cost per copy of a microarray is small, usually less than $100.@$  =   #   0(]`G [39/50& 2    0`a`4 d Advantages* 2   H  0޽h ? ̙33 (`m F(    C @h<$D 0    0n P,$D  0 KThe technique of DNA microarrays is very user-friendly: the technique is neither radioactive nor toxic the microscope slide is a convenient base for the technique arrays are cheap and easily replaced:88&      0t`G [40/50& 2    0tw`4 d Advantages* 2   H  0޽h ? ̙33)p A @8( ̙33   S  <$D 0  B  s *޽h ? ̙33 *`m  me ( s    C  <$D 0     C \`<$D 0    0`G [42/50& 2     04`4 bProblems* 2  H  0޽h ? ̙33 +PF yq (    S  <$D 0     S l`<$D 0    0`G [43/50& 2    08`4 bProblems* 2  H  0޽h ? ̙33 ,PF yq (    S P0<$D 0     S `<$D 0    0`G [44/50& 2    0`4 bProblems* 2  H  0޽h ? ̙33? -] g( w   S   <$D 0     S  <$D 0  V  0X  @`,$D  0 bdesign microarrays track clones collect, analyse and interpret data from gene expression studiescc&   Q   0`G [45/50& 2    0`4 bProblems* 2  H  0޽h ? ̙33  NA yq ( w   S 0<$D 0     S <$D 0    0<`G [46/50& 2    0(`4 bProblems* 2  H  0޽h ? ̙33.p  @8(    S  <$D 0  B  s *޽h ? ̙334 /Є   \(    S D  p<$D 0     S $ P<$D 0  A  0  ,$D 0 ADNA microarrays are commonly applied to studies involving the transcription of genes. 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O5 gHE(Ћd9cY|y !aEgK]l)a%mqe*OQ{]X$o)R#&5-Nc yd#^% 7=Č`DblG; 龛#^o|Fܹ3222,<4}^jˈɶ ,cܜS: *_ϫ"*iWRK5 [K3bߠ7jky-b^8K9c6Y|>ŢVϞ=peCCC*8cbf3#^N6׊Ζ*qsYgCHI)ֵZvSE0[=>]#dwO,%S-s%m۾opo`@&77wٲeKxha<K@ HLSN&*][,/4DQo5(]rߐiW;ڭt:̆k{*&2p.q GKPX"pI(=*#K.=RSeZv5au)DRj+w)*ߨ7`ɕ|j~{v΋F gJQ%eĊ˗/ٳ'999pIh /lBGRP vqy;/f_9mwg\:x-z;D>`%&R[էF09BJP#Rs V3!L&5*a+dPuq7ʋnw@!3rҨ~{=9;<>@aB5D FGIKYM-OQRT}VQX%Z[]_uaIcefhjmlAnpqsuew9y {|~]1هU) J Oh+'0x hp  ( 4 @LTDNA MICROARRAYS Katie AsburyYSCC:\Program Files\Microsoft Office\Templates\Blank Presentation.potDebbier30bMicrosoft PowerPointoso@@`j@@"Gg  & &&#TNPPp2OMi & TNPP &&TNPP    - "-- !-- "-&G& - &Gy& --!xH-- @Times New Roman- . 2 5uDNAMMM.@Times New Roman- .2 W MICROARRAYSb(KKQKKKLK:.--"System-&TNPP & ՜.+,D՜.+,$    ZOn-screen Show Katie Asburyw11 4Times New RomanBlank PresentationMicrosoft Clip GalleryDNA MICROARRAYS Introduction to DNA MicroarraysPowerPoint Presentation.The main applications of microarrays include:/Background to the Technique of DNA MicroarraysPowerPoint PresentationAs can be seen in the above diagram, there are four different bases (A, G, C, T) and the hydrogen bonds between the complementary pairs of bases - shown below in blue - bind the DNA strand together.HybridisationMicroarray Fabrication%Components of Microarray FabricationPreparation of SamplesPowerPoint PresentationConstruction of the ArraysPowerPoint PresentationMicrospotting TechniquesPiezoelectric PrintingPhotolithographyPreparation of the ProbesPowerPoint PresentationPowerPoint PresentationHybridisationPowerPoint PresentationMicroarray Equipment Arrayers ScannersPowerPoint Presentation Scanners commonly used include:Data Analysis and ManagementPowerPoint PresentationOligonucleotide ArraysPowerPoint PresentationPowerPoint PresentationUses of Oligonucleotide Arrays Applications of DNA MicroarraysIn Arabidopsis thaliana In Humans In YeastAdvantages of DNA MicroarraysPowerPoint PresentationPowerPoint PresentationProblems of DNA MicroarraysArray Fabrication"Equipment and the Associated CostLimitations of EquipmentData ProcessingEthical IssuesFuture PossibilitiesFingerprintsDNA MICROARRAYS  Fonts UsedDesign TemplateEmbedded OLE Servers Slide Titles1 8@ _PID_HLINKSA` http://nasc.nott.ac.uk/students http://nasc.nott.ac.uk/students http://nasc.nott.ac.uk/students http://nasc.nott.ac.uk/students,http://nasc.nott.ac.uk/students/microarrays_RDebbieDebbie  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~Root EntrydO)PicturesCurrent UserSummaryInformation(PowerPoint Document(vDocumentSummaryInformation8