《《植物质体PCR协议》.pdf》由会员分享，可在线阅读，更多相关《《植物质体PCR协议》.pdf（8页珍藏版）》请在taowenge.com淘文阁网|工程机械CAD图纸|机械工程制图|CAD装配图下载|SolidWorks_CaTia_CAD_UG_PROE_设计图分享下载上搜索。
1、Extraction and PCR of plastid DNA from a plantCharles Hill,Wymondham College,Norwich,UKLevel of difficultyAlthough this is not an easy experiment to be performed at school,it can be organisedwith help from scientists or other experienced teachers.If you need support inidentifying a scientist or an e
2、xperienced teacher to contact in your area,please ask us,sending an email to scisocembo.org.PrefaceThe D1 protein in the Photosystem II Reaction Centre in plant chloroplasts plays a keyrole in electron transport,and the structure and function of Photosystem II has beenreviewed(Hankamer,Barber and Bo
3、ekma,1997,and Raghavendra,2000).It has beenknown for some time that the site of action of certain herbicides is this D1 protein(seeDe Prado,and Jorrn and Garca-Torres,1997).Hirschberg and MacIntosh(1983)analysed the sequence of the plastid psbA gene in Amaranthus hybridus and found thatresistance wa
4、s due to a point mutation in codon 264 causing the amino acid serine to bereplaced by glycine.This was due to the substitution of the base A by G at position 790.This mutation has also been found in Solanum nigrum(Goloubinoff,Edelman,andHallick,1984)and Senecio vulgaris(Frey,1999),and has been assoc
5、iated with resistanceto the triazine herbicides.DCMU(Diuron)is also known to inhibit electron transportby binding to the D1 protein.This point mutation(SNP)creates a restriction site for therestriction enzyme BstX 1.IntroductionThis protocol provides a means of identifying either the resistant or th
6、e herbicidesusceptible genotype of Rapid Cycling Brassicas by amplifying a 963 basepair(bp)fragment of the psbA gene(from the coding region)and subsequent incubation with therestriction enzyme BstX 1,which cleaves the gene for resistance into 764bp and 199bpfragments but has no effect on the non-res
7、istance gene.The protocol is divided into 4 stages:extraction,PCR,restriction digest,and gelelectrophoresis.Certain aspects of this protocol have been designed to match aprotocol for the amplification of human mitochondrial DNA(Schollar and Harrison,2002),and readers may find reference to this proto
8、col helpful.If a restriction digest has been carried out,it should be possible to see a differencebetween the uncut fragment(963bp)and the larger fragment resulting from thedigestion of the resistant gene product(764bp).It may not be possible to see thesmaller fragment due to the sensitivity of the
9、staining procedure.If bromophenol blue isused as the loading dye then its position on the gel after electrophoresis and the smallamount of diffusion which occurs,may mask the smaller fragment.Apparatus needed(for reagents,see experimental protocol that follows)micropipettes to measure volumes from 1
10、 to 50 l,and tips(e.g.Gilson orEppendorf pipettes)plastic reaction tubes,500 l and 1,5 ml sizes microcentrifuge suitable for these tubes PCR thermal cycler(heating block with variable programme)or constant temperaturewater baths agarose gel apparatus(tank)high voltage power supply capable of deliver
11、ing up to 300 volts D.C.(a lower voltage supply can be used with longer running times)Sources of Biological MaterialSeeds for Rapid Cycling Brassicas can be obtained from the Crucifer GeneticsCooperative at the University of Wisconsin in the USAhttp:/www.fastplants.org/research/phenotype.html.Seeds
12、can also be obtained inthe UK from Blades Biological http:/www.blades-bio.co.uk although they may nothold the atrazine resistant biotype in stock.A convenient source of leaf tissue for PCR only is salad rape(Brassica napus).Seneciovulgaris and lettuce(Lactuca)have also given positive results but the
13、 primers do notappear to work with barley(Hordeum spp).Analyses of the psbA gene sequences from other plants suggest that Nicotiana tabacum,Solanum nigrum,Arabidopsis thaliana,Chenopodium rubrum and Amaranthus hybridusshould all give a PCR product using the same primer sequences.1st stage:High pH ex
14、traction method for Plastid DNAMaterial:Forceps Cocktail stick or toothpick at least 6cm long 1.5 ml microcentrifuge tube Plastic drinking straw cut into approx 5cm lengths Micropestle*or glass rod to fit tube Micropipette to measure 50l with tips 70%ethanol for sterilisation Plastic gloves Leaf mat
15、erial the cotyledon leaves of rape(Brassica napus)or other relatedBrassicas work wellSolution A 50l 0.9M KCl 0.1M KOH10mM EDTASolution B50l 0.9M KCl0.1M HCl 10mM EDTA 0.2M Tris pH 8.8If in doubt about the cleanliness of forceps or micropestle,rinse with 70%ethanol anddry.The micropestles and cocktai
16、l sticks/toothpicks could be autoclaved.Procedure:1.Pick a healthy leaf with the forceps.Use the straw to punch a small disk about2mm2(=1.4 x 1.4 mm or 1.6 mm diameter).It is helpful to support the disk ona soft surface e.g.a plastic glove on a finger tip.Use the stick to push the leafdisk into the
17、bottom of the 1.5ml tube.2.Add 50l solution A and homogenise with the micropestle but proceed carefullyto ensure homogenisation is complete and that the micropestle does not pushliquid out of the tube if pushed down too quickly.A clear green solution shouldbe the result3.Add 50l solution B with the
18、micropestle still in the tube,remove themicropestle,close the top and mix by flicking the base of the microcentrifugetube two or three times.4.Place the tube in the fridge until required Dilute the extract 15x with sterilewater(e.g.10 l extract+140 l)water.Use 15 l of diluted extract per 25 lPCR rea
19、ction mix.(The extract will keep for at least a week in a fridge)The method is a modification of methods used in the following references:P Matthews et al,(2001)Molecular Breeding 7(3)195 202D Thompson&R Henry(1995)BioTechniques 19(3)394 400*Autoclavable,reuseable micropestles obtained from Anachem
20、K749520-00002nd stage:Polymerase Chain Reaction(PCR)Amplification of the plastid geneusing PCRMaterial:DNA-containing extract from plantsReady-To-Go PCR bead(Amersham Biosciences puReTaq Cat No.27-9559-01,supplied as pack of 96 0.2 ml tubes,but I have also used BioTaq polymerase(at 0.05U per l)from
21、Bioline with KCl buffer(BIO-21040+BIO-37027),plusdNTPs at a final concentration of 0.25mM each.Primers:purchased from Invitrogen using 50 nmol synthesis:P1=5-CGAAAGCGAAAGCCTATGGG-3P2=5-TCCATACCAAGGTTAGCACGG-3Primers are dissolved in TE buffer to provide a stock solution of 1 g/l(approx.150M).Procedu
22、re:1.Before use,primers are diluted 30 times with DNA-free water(e.g.5 l primer+145 l water).If volumes smaller than 10 l cannot be easily pipetted,thenequal volumes of the two primers might be mixed and 10 l of the mixtureadded.In this case primers should be mixed immediately before adding to thePC
23、R tube and kept on ice once mixed.2.Set up the PCR reaction mix.The total volume in the tube is 25 l.Thisincludes:15 l diluted extract or 14 l of DNA free water+1 l of original DNA extract(add water first)5 l of Forward Primer P1 (Final concentration=1 M)5 l of Reverse Primer P2 (Final concentration
24、=1 M)3.PCR reaction.The following programme is used in the PCR machine:1.94C for 2 minutes(initial denaturation)2.Denature -94C for 30 s3.Anneal -55C for 30 s 30 cycles4.Extend -72C for 30 s5.72C for 5 minutes6.Hold at 4CKeep the tubes on ice until the restriction enzyme digest.3rd stage:Restriction
25、 enzyme digest of the PCR productReagents:PCR productRestriction enzyme:BstX I,1000 units,New England BioLabs(NEB)R0113SBuffer:NEB Buffer 3(supplied with the enzyme)Procedure:Carry out for PCR product of both Atrazine Resistant and Sensitive plant extracts.1.Add the following to a microcentrifuge tu
26、be(approx.50 ml total volume):25 ml PCR product20 ml DNA-free water5 ml Buffer X101-2 ml BstX I enzyme(10-20 units)2.incubate at 50C for 1hPut samples on ice until the next stepUse at least 30 ml of digest per well for electrophoresis check that the wells of the gelhave the right size.4th stage:Gel
27、Electrophoresis and StainingReagents:PCR-product after restriction digest(use at least 30 l)Gel:1%agarose in 0.5 X TBE Buffer with wells 5.5mm x 1.5mmLoading dye:Orange G(acid orange)at 6x final concentration:25 mg dye+3 mlglycerol+7 ml water(or instead of glycerol use 4g sucrose in final volume of
28、10 ml water)Buffer:TBE=90 mM Tris base,90mM Boric acid,2mM EDTA,pH 8.3Staining solution:0.1%methylene blueProcedure:1.Remove the tube from ice.2.Set the micropipette to 5l and,using a clean tip for each PCR tube,load 5l ofloading dye(LD)(Orange G in a glycerol solution).This colours the samples andm
29、akes them denser than the electrophoresis buffer.3.Add the loading dye to each tube(and,if several tubes are to be done,eject thetip into the tube the tip can be used again to load the sample onto the gel see below).Make sure that the loading dye is deposited near the contents at thebottom of the tu
30、be.4.The gel is in a tank with electrophoresis buffer covering it.Each participantshould be able to load at least one well.Make sure that you label the gel map.5.Reset the micropipette to 20l.For each tube mix the contents by pipetting upand down,and then load the sample into a well on the gel.6.Whe
31、n loading the gel ensure that the pipette tip is below the surface of thebuffer but just above the well.The loading buffer will allow the sample to sinkinto the well but depress the plunger slowly while adding the sample to the well.7.The gels can be run for about 20 minutes at 100V(the optimum is 5
32、V per cmbetween electrodes).8.When the electrophoresis is finished,the buffer must be poured off carefully,using a gloved hand to keep the gel in position.9.The gel is then removed in its carrier and added to a plastic tray containing 0.1%methylene blue.It should be left in the tray for 3 minutes wi
33、th gentle movementnow and again to aid the staining of the gel.10.After 3 minutes pour off the methylene blue stain and add water to cover thegel well.11.Leave for 10 15 minutes;if possible change the water once during this period.Destaining may be speeded up slightly by using a steady but gentle st
34、ream ofwater direct from the tap.Safety WarningIf using voltages above 25V,then designated equipment must be used so that liveterminals are properly shielded.Alternative staining:As an alternative to the staining of the gel with methylene blue,Nile Blue sulphate canbe dissolved in the TBE buffer(1g/
35、ml)used to make the agarose gel and in the runningbuffer.This stain attaches to the DNA as it migrates through the gel and results in amuch lower level of background staining,and the DNA bands stain during the actualelectrophoresis.With this method gels are best observed immediately as the stain may
36、fade with time.ReferencesWeed and Crop Resistance to Herbicides(1997)Ed.De Prado,R,Jorrn,J and Garca-Torrres L(Kluwer Academic Publishers ISBN 0 7923 4581 9)Frey J(1999)Genetic Flexibility of Plant Chloroplasts Nature 398 115-116Goloubinoff,P,Edelman,and Hallick,R(1984)Chloroplast coded resistance i
37、n Solanumnigrum:psbA loci from susceptible and resistant biotypes are isogenic except for a singlecodon change.Nucleic Acids Research 12(24)9489-9496Hankamer,B,Barber,J and Boekema,E,(1997)Structure and Membrane Organisationof Photosystem II in Green Plants.Annual Review of Plant Physiology and Plan
38、tMolecular Biology 48 641-671Hirschberg,J and McIntosh,L(1983)Molecular basis of herbicide resistance inAmaranthus hybridus.Science 222 1346-1348Photosytnhesis:A Comprehensive Treatise(2000)Ed.Raghavendra A.S.,CambridgeUniversity Press ISBN 0 521 78444 1(esp Chs 2 and 3)Schollar,J and Harrison,A(200
39、2)Amplification of Human Mitochondrial DNABioscience explained 1(2)1-10 AcknowledgementsCharles Hill gratefully acknowledges the support given through Science and Plants forSchools(SAPS)/Robinson College,Cambridge School Teacher Fellowship and thesubsequent support of SAPS and the John Innes Centre,
40、Norwich,UK for the final stages ofthe development of this protocol.Diagram 1:Extraction and PCR1.Cut disk from leaf with strawXTake care!2.Use woodentoothpick to pushdisk to bottom ofmicrocentrifugetube.(tube 1)3.Add 50 l of solution A 4.Grind leaf disk with minipestle.(If the mini pestleis pushed i
41、n with toomuch force you may losecontents)6.Dilute the extract 15 times by adding 10 l of the extract to 140 l of sterile water in a cleanmicrocentrifuge tube (tube 2)(140=20 x 7 or 50+50+20+20)7.Add 15 l of this diluted extract to the bead in the PCR tube.8.After adding the DNA extract,add 5 l of each primer(P1 and P2)(total volume in PCR tube is 25 l)5.Add 50 l of solution Band use mini pestle to mix.Remove mini pestle.Diagram 2:Electrophoresis1.Add 5 l loading dyeto PCR tube and mix2.Remove 20 l of themixture5 l25 l20 l well tip buffer gel 3.Load well with20 l