oligo 7 使用介绍.pptx

1、Oligo 7Primer Analysis Software,Rules for PCR Primer Selection and Presentation of the Software,2010 Molecular Biology Insights,Inc.,Contents,1.PCR Primer Selection Criteriaexplanation of primer stability calculationswhat makes a good PCR primerother factors besides individual primer characteristics

2、 influencing PCR reaction2.Oligo 7 Analyze Featuresthe Sequence windowgeneral information(the Key Info)duplex and hairpin formationdata about primers(Composition and Melting Temperatures)false priming sites and homology analysisoligonucleotide stability graph(Tm,DG)internal stability and its importa

3、ncesequence frequencyother DNA analysis options:ORF,restriction sites,DNA calculator3.Search Optionssearch for primers and probesother searchesbatch processing4.Oligonucleotide Databasemultiplexing5.Concluding Remarks,Primer Stability CalculationsAn introduction to the nearest neighbor method,One of

4、 the most important characteristics of a primer is its the melting temperature and stability of various its regions.The most accurate Tm calculation provides the nearest neighbor method.The primer Tm formula is quite complex:Tm(C)=DH/(DS+R*lnC)+16.6 log K+/(1+0.7*K+)273.15where DH and DS are the ent

5、halpy and entropy for helix formation,respectively,R is the molar gas constant(1.987 cal/C*mol),and C is concentration of the primer/probe(that is always used in several-fold excess than the target DNA),but fortunately computer does all the work.To understand complexity of duplex stability calculati

6、ons it is easier to explain it by showing how free energy(DG,another measure of DNA helix stability),as the formula is simpler:DG=DH TDS(T is the temperature in K).Although is looks simple enough,it is not so simple if you actually do it.Heres the example of DG calculation of a 4-mer TCGA,hybridizin

7、g to a longer strand like this:,Now,the DG can be calculated like this surprisingly long formula:DG(TC)+DG(CG)+DG(GA)+initiation DG for T+initiation DG for A+DG of 3-dangling end AC+DG of 5-dangling end GT The next slide shows the calculation graphically(data for temperature of 37).,DG calculation e

8、xample,Total DG=1+1+0.3+0.5-1.3-1.3-2.2=-2.0 kcal/molThis is a rare case,as usually the dangling ends are stabilizing.This is shown to alert you that sometimes adding a base may actually decrease melting temperature of a duplex.The next slide shows an example where adding even 2 bases has no effect

9、on theTm.,Here is a screen shot showing the selected primers and probes:,The Forward Primer Tm is 56.7,while the Reverse Primer Tm is 56.2(not much different).By adding one base on each end,and selecting the fragments as Upper and Lower Oligos,we can see that the Upper Oligo Tm is 56.7(no change in

10、Tm)while the Lower Oligos Tm rose up to 61(from 56.2).Without consideration of the dangling ends,increasing primer length always increases its Tm.More comprehensive analysis data for those particular oligos you will see on the Key Info window explanation.,What makes a good primer,High priming effici

11、encyto achieve this,a good primer should beof a reasonably high Tm,without dimers,especially on their 3-ends(to prevent self-extension),without hairpin stems,especially on their 3-ends(to prevent self-priming),lacking repetitive sequences to ensure quick and correct annealing.all primers/probes in o

12、ne incubation mixture should not form significant 3-dimers between each other.High specificityto achieve this,a good primer should belong enough to increase specificity,unique,especially at its 3-end,to avoid false priming,moderately stable at its 3-end(as opposed to highly GC-rich)to ensure that a

13、very short fragment wont initialize the extension(too low 3-end stability hurts the priming efficiency).,Other factors besides individual primer characteristics influencing PCR reaction,Besides the primer properties listed on the previous page,PCR efficiency also depends on the template.Regions with

14、 high secondary structures and GC islands amplify poorly.Oligo 7 penalizes primers that would amplify strong hairpin structures,decreasing overall scores for PCR primer sets.If you have to make hard to amplify products,select primers with high scores(usually with higher Tm and GC contents than stand

15、ard),and adjust composition of your PCR mix by adding solvents,additives or reducing/changing salts.Increasing primer concentration above 1 mmol is not recommended due to unspecific primer/primer extensions(even if the software does not show any 3-dimers).,Oligo 7 Analyze Features:the Sequence Windo

16、w,The Sequence Window is the central window that shows several features of the sequence and selected primers.When you open the test sequence,provided with the software,you will see this screen:,The sequence file name is in the top left.In the top you see also 3 tables.The first on the left shows the

17、 sequence length,the reading frame for the translation(protein sequence is a colorful string of letters,at the bottom),the Current Oligo(sequence surrounded by a grey rectangle)length and position,and its Tm.The second table lists positions of selected primers,oligos(to select,click the square locat

18、ed on the left of the oligo name),and the PCR product(for this,you need to select 2 Primers).Click the“i”to get more info about the selected Oligo.The third table lists sequence features(if sequence annotations/features are present;you may enter them manually from the Edit Menu).,Oligo 7 Analyze Fea

19、tures:the Sequence Window,Below the tables you see the oligonucleotide stability graph(default is Tm).It is a miniaturized Melting Temperature Graph Window,available from the Analyze Menu.Below the Tm graph there are a series of horizontal lines or rows.Each row may contain a graphical representatio

20、n of various features spread out in the entire sequence,such as primers positions,PCR product and others.In this example,the first filled row is a pattern of red rectangles symbolizing weak hairpin loops in yet to be selected primers.The green rectangles below the red row represent palindromes.The l

21、ast filled row shows the coding sequence as a blue line,as the Feature Table highlights“CDS”.,The bottom part of the window shows the enlarged part of the sequence,that is highlighted in yellow in the Tm graph(top left of this picture).The cursor position and the Tm of an oligo that would start at t

22、he cursor position is indicated on the right,just above the position numbers scale.Below the scale you see the DNA sequence,where the positive strand is in red.If you select primers they would appear above and below the sequence.Translated protein sequence is displayed in various colors.The colors s

23、ymbolize codon frequency(black is the most frequent,see the Open Reading Frames window).The rows below and their colors symbolize identical features,but enlarged,to those displayed just below the Tm graph.More detailed explanation is on the next slide.,The graph in colored rectangle is expanded in t

24、he zoom area,With Oligo you can select up to 4 primers or probes,Explanation of other symbols in the Sequence Window,The Key Info Windows,The Key Info windows display basic information about selected primers or probes.These are the oligos discussed in the previous section,where a longer 13-mer oligo

25、(lower left)has the same Tm as its shorter 11-mer version,displayed above it.The tm is a melting temperature calculated without the dangling ends effect,and this one increased by almost 7.Note that the complementary oligos of the same length,shown on the right,have the same tm as expected,but differ

26、ent Tm,especially the 13-mers.,More statistics on those oligos provide the Composition&Tm windows,as well as the Concentrations window.,The Duplex Formation Windows,The Duplex Formation windows display the strongest 3-duplexes,the most stable duplex that is not necessarily at the 3-end,and the stron

27、gest hairpin.The DG data are given with consideration of the dangling ends and initiation DG values.The Mixed Oligos Duplexes window does not display hairpins.,The Hairpin Formation Windows,The Hairpin Formation windows display all possible hairpins in a given oligo,listed from the strongest one to

28、the weakest.When a given segment is involved in many different structures,the real melting temperature of a particular hairpin is lower than calculated due to competition between the structures.,The Composition&Tm Windows,The Composition&Tm windows display melting temperatures calculated with differ

29、ent methods widely available,that is the nearest neighbor method(Td is a normalized Tm in 1M salt conditions,while the Tm is a variable value,that depends on salt and DNA concentration set in the Search Parameters window).The Tm Calculated with the%GC method do not depend on the Search Parameters se

30、ttings.Also,the simplest method of Tm calculation(2 for A or T and 4 for C or G)is given,followed by the Tm values of RNA and DNA/RNA hybrids.The second table lists base composition,and the last the Tm values in various salts and formamide concentrations calculated with the nearest neighbor method.,

31、The False Priming Sites Windows,The False Priming Sites windows display potential false priming sites of primers.This particular example shows an old commercially available universal m13 reverse primer.The site with P.E.of 244 points is the only real false priming site as confirmed experimentally(St

32、effens,D.L.,Sutter,S.L.,and Roemer,S.C.(1993)“An Alternate Universal Forward Primer for Improved Automated DNA Sequencing of M13”,BioTechniques 15,580-582.),The Homology Windows,The Homology windows display homological sites for probes,and lists the sites from the highest similarity to the lowest.Th

33、e 3-end is irrelevant in this case,and homology of a probe(or a primer)is aligned with the actual DNA strand,and not with its complement,as in the False Priming Sites window.When using shorter DNA fragments in hybridization experiments,a homology below 90%is usually not significant,and wont produce false signals.,The Selected Oligonucleotides Window,After a search for Primers&Probes you may open t