High Resolution Melt Analysis (HRM) and Its Strategic Applications Especially in Molecular Genetics

©2016 ASP Ins., Afarand Scholarly Publishing Institute, Iran ISSN: 2252-0805 Quarterly of the Horizon of Medical Sciences 2016;22(1):77-88 High Resolution Melt Analysis (HRM) and its Strategic Applications Especially in Molecular Genetics

A R T I C L E I N F O A B S T R A C T Introduction In wide and developing worlds of cellular and particularly molecular sciences, Article Type the most important issue that leads to paying more attention to and use a technique, is its Analytic Review Resolution Melting (HRM), may be one of these techniques. Its usage is growing among Authors critical features; high sensivity, specificity, velocity and also being available and cheap. High Noori-Daloii M.R.* PhD, and characteristic of DNA when melting. DNA melting temperature (Tm), is the temperature Faraji K.1 BSc thatothers 50% significantly of DNA molecules in investigations, have become for its specialsingle stranded advantages. and HRM 50% is are based double on the stranded. pattern

How to cite this article differences are shown as a graph called “melting curve”. These are effective on DNA Tm: the Noori-Daloii M.R, Faraji K. High valueThe pattern of bases of G gettingand C in single both strandedDNA strands is specific (CG%), andthe length unique of for fragment, every DNA the static strand; effects and Resolution Melt Analysis (HRM) of bases on each strand and the hetrozygocity. In fact, the differences among melting curves and its Strategic Applications Especially in Molecular Genetics. results in recognition the special fragment. The way of being single stranded can be followed Quarterly of the Horizon of Medical Sciences. 2016;22(1):77-88. Conclusion High-resolution melting is powerful, fast, comprehensive and useful technique forby fleurcenceusing in molecular colors which laboratory attached that to double can be stranded considered DNAs. as simple and fast approach for genotyping, mutation detection, matching sequence and methylation study.

Keywords High Resolution Melting; Molecular Biology; Molecular Diagnostic Techniques

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Copyright© 2016 ASP Ins. This open-access article is published under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License which permits Share (copy and redistribute the material in any medium or format) and Adapt (remix, transform, and build upon the material) under the Attribution-NonCommercial terms. 0 0, 1( 2 #* ' 0( ./ (, -( DNA . 789!' 5 (3 *4 #( 1( @ .($ ( (= +-$ >;< ) HRM #* CDDE #B ( ' 0* 2 9A * )9K 8' + F)( G$ H) IJ PhD F( &$ ./ 5( *M L I=( .[1, 2]0( ( DNA ;< * N )9K 9' #* #( ' .(8 BSc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a4`$ 9@7P _$Y #M$ ^$K9 9H] @ . G - 6 &' 6@6 A% c7^$HLA 9b"& DNA )')N (SNP) "6$ ( 6+ 8 .%$ % DNA .% [1-7] 5( #O9 . d ./ (. F( .B D % DNA .(7 #* #( .(7P (7/ ' 7bK 6B$- < ? ,)$ <9: : 9F)1/ 4K A + / 9 8A*3/ 1 % ) ' . (, -( +Y $ #$ % < % % ./ .%$ ( 0O9( 1( (O ( N 90- "&( .2% . A% %$ $ "' e fB)$ ' 0( \( #7P .0( #* #( &$ R, *+, -. /(/0 *1-"#$ %& '() & *! : . 9(3 g %7 ( 8Q$ N 0( h */(/0 1 8Q$ ( O 0( 1 4 [(Y( G( #7iO9 799/78/45 :".2 32 3 HRM #* _$Y #M$ e ( %7 795/85/7 : 32 [8, 9] * . 0( "/ F(& [email protected] :#0 -#

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Q Horizon Med Sci Vol. 22, Iss. 1, Win 2016 HRM A 1( k(3( .(| R) 9(3 I- e& 7L7 1( 97P #( 8O$ HRM ($ ' 0( '< 1= BRAF .iO9 9.Y \N 9b"& ( G$ | 97P fB)$ .[11, 19]0( I- F 0, 0 1( ' 0( -( KRAS PIK3CA F #( fB)$ ( .0( ( '( HRM .[23-27]0( (3 l, R, &D/U IJ ( b(8*( ' ( ( 0, #( fB)$ ( T A 9$a' .( $ O O( &D/U [8, 0( 91 `$(@( I-$ .($ ( #( 0 ( .20] 1( k O N 9 O _$Y #M$ ( HRM A ;K 1 #(7 -( PCR 9/* !F 2 ($ ` ( A>C X$ ( O ( .0 A/C IJ !9 &( ./ 4 9_$Y 4 0 A/A 90 ( 7L7 F( . 9(3 C/C A/A R 1( $= &` C/C R 9(3 .@ C/C > !& .> 0 +B% HI &AB "F (" I- !9 8 IF19 9* F( . (, A/A DNA HI &AB "F E& DNA " 0A .(E R) 9(3 .&J

.( 8/ .K 1( [K 9O .! R HRM #* .( & "/ F .7* 1( -( -( ~@) HRM /0 d i7P O ( 1( $ O ( ($ ( ( 9O $ ( .($ .[1-3, 23] #aO\ HRM d F N 9b"& ( HRM #* 1( -( .0( (3 = 0, 0 1( 9A (DHPLC DGGE SSCP 7) (1( e 9A , DNA &AB /@ (! h(( Fm ( IJ ' ( & HI *$G$ &AB "F E& D$C + 0A 1( 9A #( . Q( PCR 9/* 9b"& .&* ' -( DNA 77'0( + [$ .[6, 28, 29]0 R( #( 1 HRM #* O () !O ;B( $ e ( I(X$ 1 ( 9M\, (( +$ 3 @7P) SSCP A ( !27$ D/U #( &+ IJ b(8*( ( ( DNA [^ .7 $ PCR +O' DNA [@KO9 (C/A A/C) [@K9 90 .($ 1(( DNA 0' . ( 0' 1*( Y F( 4 0( 4< 1= .' 7 8 ( (C/C A/A) Y u*7 1( +P' 9 4 L ( F M\, v 7L7 F( "Q _$Y ` v 97L7 + 1( (( SSCP 1*( .77' l / Q7 (A/A O () fB) (( _$Y ` ( DNA Rl$ ( ( 7 77'0( 9( 8/ L . ( fB)$ "Q _$Y 2 M\, )"& ( . -( ( +$ .[21, 22]0( * (, }L (( HRM 1( RJ 9(3 I- 1*( "/ 0' F( ( & %& $ HRM (# 9M\, 1( ( b"& (( 9M\, .($ %$$ #( " ' ' ( y\ A #( .( 8O$ MlZ #M$ 7O9 b"& 7 #M$ ( HRM A ' 0( ./ A #( O R) P ( . * #( . -( PCR I=TL 1( kON _$Y t$ "/ 1( (1 O 7 9b"& 1( 7L7 $ 0( 1 (( e& 7L7 + 8 A 9M\, I&" F( $-$ Q ( b"& (( IJ . Q7 ./ "Q O 9O 1( RJ y\ 5( #O9 .O 9) Y ./ 1( RJ 7L7 2 X$ h 9 & DNA &51 .'K)&!5 51&@$ DNA ' .[21]%e DNA % Ye! .@& 6C 6!& . .I'1P %&'! .[4, 6, 22]! MS- 7< 5` .^ ! ^ = .&51 K'! (,,-! " #$ %&'()) DGGE 7 .951 7. .I1 J1 W/ HRM "1. 2 34 SSCP ./01 >@ % 7< 5` .^ ! ,< W! ;< =9 >?@ 601 1 7@51 678 19- :&1 X &`. .^ Methyl Primer Express® v1.0 . ;< B'C/@ 3? . %@ & .[11]& K'! ! .1@ .I'1 7< 5` DNA . EF4 .- 5&D .5 1 >?@ PCR 9 @ =.I'1 I4 .^ 5! E =&(Ie19 #&` LF! @9 = ! , 3K'1 J &HI-&@ G .^ ,'/ 5b@ .I7 % 7< 5)& = CpG 4 =&'! DNA .@&7@ .51 M 5K'1 E .U B'EC` .@ 4 .& &1 7< 5` 1 OIP % 5'C/@ .- 196- 9N I< #$ G % .@ ' O@ &1 .EF4 @ =.I'1 I4 .^ 5! Q #$ @51 4 ! (11 B2&7E1) ,,- ! Bc 1 ^ .I'1P V1 .@&7@ =9 Tm fc'] =1 1 OIP % 5C!,1 .F0@ . .- 5@1% ,,-51 - . RS 5,g,1 ' 7 "&1 1 .&51 ' .I'1 .F0@ .,7@51 = . Q ,!51 ,,-! % .^ [U 3201 C'E1 .&51 #&?1 .@&7@ 3&D. ) - 678 RS .F0@ G =&,8. =&(Ie19 #&` ! K'! MS-HRM .EF4 &51 J- .EF4 - 8! % T7U & ! 2 @&.I'1 ! E %K .EF4 " . .! 3K'1 .EF4 J)! .[21, 35, 36]! .[12]&51 5 ! %@ &1 =.I'1 !/ h- .- 51T, ' (2 59- DNA V1 5&1-) DHPLC @ .^ ^ 5` &` 7< =& 51 K'! &HI-&@G 5I(,U 5!, % .@&7@ =&7@.'K)&! 5 % Y< 5C8. ., CpG >?@ PCR 9 ! 2 34 >T, =9 #C@. @&51 6C 6!& . .I'1P %&'! .- ! DGGE .1 BC0 W! .&51 .I'1P jDNA . CpG 7< #i Y(I<' #&()&1 . 4 =TX< =1 1 7 .- &] & "% &@ =K " .[12],-51 PCR )& 6(. .I'1 .EF4 .?'@ .51 - 7< 5% 51 2 &1 ./01 HRM ,;7 . ,&] hb )&!. .' #i 7< . 61^ :[1-3, 12] % &1 67'1 =9 5X & 4 7< 3' .@ 5b@ CpG .;@,U .Y(1 . %@ >8 (Z) • .[21, 35-38] &] .I'1 DNA . #i .,,-! 1 . %@ >8 (R • #&?1 .@&7@ % 6D RS 5,g,1 >?@! ) .)&) = HRM ,- .7 >?@ ([ • V1 .@&7@ . k&1 RS 5,g,1) @'! RS 5,g,1 9 %! \] . %@ >8 (.'/ .)&) ./01 (,'/ 6'1 1 3K'1 D .- .PCR .&51 .?'@ Ye! ,^ . 5&)9 #7' =!64 . (3 • ..)&) % 9 \] >8 6). 5 HRM !/ 5X .- ! -S . >%2 HRM %K #&` PCR 9 &HI-&@G 3_ O@ &1 .EF4 #&` R'@ C'E1 57I8 V,1 W! %K #&` PCR 9 :[2-4, 20, 39]& 8 % &1 h( [1, 2, ! / =9 5X %/ =9 !/ :&' &$)* $ (% .26, 30-34] SNP &l #7' % % ' .EF4 #&` HRM ( 5,g,1 .?'@ .' O@ &1 .@ .'!&]@ 5ID ^%! % 5b DNA =.I'1 &T) 3_ #7' .EF4 #&` ,;7 .&51 ;< 6D HRM) MS-HRM .! =$ = #',^ 5b'@$5< 51 .' DNA .&@: ']! 6( @& 51 .^ ! ! / d (=&!c'1 . W/ . ,&] 5K'1 RS &T) 3K'1 ']! .F!. .- >&@$ =.I'1 &T) 5! . 5^ 8! 4

Q Horizon Med Sci Vol. 22, Iss. 1, Win 2016 Edit ., R'@ >U .I1 >?@ 5< 6( mg) % O@ &1 .@ R'@ % Y< &51 .D& .^ J&51 PCR >?@ q . k&1 7/4 Profile ! % &O,1 .& 5! @ .&@: ']! J@9 =#E >%2 =1%31 T@@ Hold ., ?, O@ &1 5)& .- &g@ . & K'! DINAMELT Ye! .&51 JO, °C .04 Bh- .- ! %15I< .& R'@ Submit ., &7@ E .@ 1 .]U E =! 1 Cycling ., >- 6( ΔG =1 7. .&@: ']! 6( @ .&51 JO, 7< #i .&51 7@ ) 51 % Hi-Res Melt .,% K'! =9 5< 6( ΔG #&C4 64 =1 .- ! -S . >%2 (. /) 1 =1 O@ &1 ( °C .@&7@ .! .&@: ']! 8 Gain Optimization 7/4 &51 R'@ n@ #i =1 &HI-&@ % '7- .EF4 #&` .;@,U 01 % Y,!&o&I &@ 3 .- 5,E1 . r&51 R'@ n@ 3_ C: 5 @ & J- Y,!&o&I .! 5!, 64 .& &] 6(1 Y,!&o&I #',- j 38c` =197@ . :345 *. + , - . & ( PCR ,9 Start Run ., R'@ @ . :[2-4, 20, 39]' .) &51 Q HRM SNP C@ &51 5` =9 7< .- .@ .(& .E1 jcu.edu.au/cgc/HRMTA_design.pdf .&51 ;< 5,g,1 ? "&1 % . &HI-&@ 7< #&` HRM 0$6 Primer Express® Primer3 ,@1 5>@ % :& 5` % p. .I1 5I^ ) HRM )@9 .& K'! 7< 5` 3.0 =&"!,1 gD E &O,1 . :( 6b) hb .I1 . °C °C (Tm) 7< #i 1 5,g,1 .51 4 5! &1 hb 5,g,1 hb °C % ' < W&(E1 7< (Tm)1 fc'] h( @ #c] .- 3&D . ! &ED 3&D. .C@ . Y,!&o&I n@ #i =1 PCR 9 % . % 7< GC % =1 @ Y,!&o&I Rt .' .' 9 % ' 01 . 7< 3' '@ ΔG 3b@ HRM )@9 .^ ! ^S = .51 @ - 7< 3' '@ #i #7' 5,g,1 "5%" .b@9 X. r 4 .& &1 571 #7' @ % '7- 01 . &@ )& 9 .ID ! 5)&C4 64 "% .I1" . / 2 .51 5Di']P 9 )& C =1 .[1, 2, 11, 40]C@ .]U % ' .@&7@ C T T . h- 7 7< 6( ΔG 9 OIP =&3K'1 T@ 3K'1 J .@&7@ .C@ .! % ' 5' '@ CG % E &] /K =;< "&1 1 7 .- ! PCR 5K,1 .^ %@ . .- ! Ye %9 $@ =7 ΔG 50'1 5,g,1 5! :( 6b) RS 5,g,1 5! .! (7< #i) ,- >?@ =&&] .&] ,=@ =&5Di'] % =,7` "/^ =9 f 51 >?@

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)G" ," /'6U )4 /r"s Y" f4- - tC ' # )!B HC+ :C% 8( MgCl2 HRM > &" .D 8-d+ FG )- .- /)#HU a 3- /QP+ )"G+ #!4 .6 !" $# )G" Y4^ #B4( \" (H ) (%) ;\R 8R /88 Y" #B4( -d @ /2LO+ Y4 f DGGE /SSCP +) @-+ #A F4 D4X+ / Q; Y4 89 .)+ ;" 6 #)>Z4 4 Y Y4 /)>G B 8-2 (DHPLC " 8")+ Y]B# . )_ 4 #B4( .t+ O+ 8 4- Y49F4U 8^ . '#e !" #R^ HI ) ]( HI #)*+ :>^ HI ) .- O A9> 8>D4 E-" k+ : >^ .- O A9> 8>D4 E-" k+ : ,_+ ]( HI k#)*+ DP" e - e" 84 .- O A9> 8>D4 E-" k+ : # )+ b )+ G Y4 .- D >^ HI .- O A9> 8>D4 E-" k+ : Y4 C3 e - G O (8!Cn+) G_ :> : 4 + 8")+ 2!O+ ) "qCem" )C!^ Y4^ (jG 1- Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods. 2010;50(4):250-61. )4)G" /2LO+ 8 2 e (8r"s 4 2- Erali M, Voelkerding KV, Wittwer CT. High resolution ]^ .- )C!^ " )- PCR melting applications for clinical laboratory medicine. Exp Mol Pathol. 2008;85(1):50-8. * #B4( D4)+ ? )C!^ Y4^

DNA Science. Switzerland: Springer International Publishing; 3- Applied Biosystems. A guide to High Resolution 2013. pp. 171-85. Melting (HRM) analysis. 2012. Available from: 20- Mann T, Humbert R, Dorschner M, http://www.gene-quantification.com/ab-hrm-guide.pdf. Stamatoyannopoulos J, Noble WS. A thermodynamic 4- Ye MH, Chen JL, Zhao GP, ZHeng MQ, Wen J. Sensitivity approach to PCR primer design. Nucleic Acids Res. and specificity of high-resolution melting analysis in 2009;37(13):e95. screening unknown SNPsand genotyping a known 21- Chen D, Wang YY, Chuai ZR, Huang JF, Wang YX, Liu mutation. Animal Sci Papers Reports. 2010;28(2):161- K, et al. High-resolution melting analysis for accurate 70. detection of BRAF mutations: a systematic review and 5- Cho MH, Ciulla D, Klanderman BJ, Raby BA, Silverman meta-analysis. Sci Rep. 2014;4:4168. EK. High-resolution melting curve analysis of genomic 22- Guedes JG, Veiga I, Rocha P, Pinto P, Pinto C, Pinheiro and whole-genome amplified DNA. Clin Chem. M, et al. High resolution melting analysis of KRAS, BRAF 2008;54(12):2055-8. and PIK3CA in KRAS exon 2 wild-type metastatic 6- Noori-Daloii MR. Medical molecular genetics in the colorectal cancer. BMC Cancer. 2013;13:169. third millennium. Tehran: Samber Publishing; 2009. 23- Gonzalez-Bosquet J, Calcei J, Wei JS, Garcia-Closas M, 7- Castellanos E, Aranaz A, De Buck J. PCR amplification Sherman ME, Hewitt S, et al. Detection of somatic and high-resolution melting curve analysis as a rapid mutations by high-resolution DNA melting (HRM) diagnostic method for genotyping members of the analysis in multiple cancers. PloS one. 2011;6(1):e14522. Mycobacterium avium–intracellulare complex. Clin 24- Pichler M, Balic M, Stadelmeyer E, Ausch C, Wild M, Microbiol Infect. 2010;16(11):1658-62. Guelly C, et al. Evaluation of high-resolution melting 8- Reed GH, Kent JO, Wittwer CT. High-resolution DNA analysis as a diagnostic tool to detect the BRAF V600E melting analysis for simple and efficient molecular mutation in colorectal tumors. J Mol Diagn. diagnostics. Pharmacogenomics. 2007;8(6):597-608. 2009;11(2):140-7. 9- Watts AM. High resolution melt analysis: a novel 25- Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A. method for studying the genetic relatedness of High resolution melting analysis for rapid and sensitive Pseudomonas aeruginosa isolates from clinical and EGFR and KRAS mutation detection in formalin fixed environmental sources [Dissertation]. Brisbane, paraffin embedded biopsies. Bio Med Cancer. Australia: Queensland University of Technology; October 2008;8:142. 2013. 26- Krypuy M, Newnham GM, Thomas DM, Conron M, 10- Noori-Daloii MR, Jalilian N. Applications of Dobrovic A. High resolution melting analysis for the comparative genomic hybridization in cancer and rapid and sensitive detection of mutations in clinical genetic disorders: A review article. Tehran Uni Med J. samples: KRAS codon 12 and 13 mutations in non-small 2011;68(1):1-11. [Persian] cell lung cancer. Bio Med Cancer. 2006;6:295. 11- QIAGEN. Principles of High Resolution Melting 27- Liu Y-P, Wu H-Y, Yang X, Xu H-Q, Chen D, Huang Q, et (HRM) technology [Cited 2015, 4 July]. Available from: al. Diagnostic accuracy of high resolution melting https://www.qiagen.com/ir/resources/technologies/hr analysis for detection of KRAS mutations: a systematic m/principle%20of%20hrm%20technology/. review and meta-analysis. Sci Rep. 2014;4:7521. 12- Noori-Daloii MR. Emery’s elements of medical 28- Li J, Wang X, Dong R, Yang Y, Zhou J, Yu C, et al. genetics. 6th editon. Tehran: Jame’e Negar and Salami Evaluation of High-Resolution Melting for Gene Mapping publishing; 2012. in Rice. Plant Mol Biol Rep. 2011;29(4):979-85. 13- Rubinstein WS. Hereditary breast cancer: 29- Wittwer CT. High-Resolution Genotyping by pathobiology, clinical translation, and potential for

Amplicon Melting Analysis Using LCGreen. Clin Chem. targeted cancer therapeutics. Fam Cancer. 2008;7(1):83-9. 2003;49(6 Pt 1):853-60. 14- Noori-Daloii MR, Ebrahimzade Vesal E. Molecular 30- Reed GH, Wittwer CT. Sensitivity and specificity of genetics, diagnosis, prevention and gene therapy in single-nucleotide polymorphism scanning by high- prostate cancer: Review article. Tehran Uni Med J. resolution melting analysis. Clin Chem. 2009;67(1):1-14. [Persian] 2004;50(10):1748-54. 15- Noori-Daloii MR, Maheronnaghsh R, Sayyah MK. 31- Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding Molecular genetics and gene therapy in esophageal KV. Expanded Instrument Comparison of Amplicon DNA cancer: A review article. Tehran Univ Med J. Melting Analysis for Mutation Scanning and Genotyping. 2011:69(6):331-43. [Persian] Clin Chem. 2007;53(8):1544-8. 16- Wang F, Shen H, Guan M, Wang Y, Feng Y, Weng X, et 32- Martino A, Mancuso T, Rossi AM. Application of high- al. High-resolution melting facilitates mutation screening resolution melting to large-scale, high-throughput SNP of rpsL gene associated with streptomycin resistance in genotyping: a comparison with the TaqMan method. J Mycobacterium tuberculosis. Microbiol Res. Biomol Screen. 2010;15(6):623-9. 2011;166(2):121-8. 33- Montgomery JL, Sanford LN, Wittwer CT. High- 17- Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding resolution DNA melting analysis in clinical research and KV. Expanded instrument comparison of amplicon DNA diagnostics. Expert Rev Mol Diagn. 2010;10(2):219-40. melting analysis for mutation scanning and genotyping. 34- Janne PA, Borras AM, Kuang Y, Rogers AM, Joshi VA, Clin Chem. 2007;53(8):1544-8. Liyanage H, et al. A rapid and sensitive enzymatic 18- Takano EA, Mitchell G, Fox SB, Dobrovic A. Rapid method for epidermal growth factor receptor mutation detection of carriers with BRCA1 and BRCA2 mutations screening. Clin Cancer Res. 2006;12(3 Pt 1):751-8. using high resolution melting analysis. Bio Med cancer. 35- Wojdacz TK. Methylation-sensitive high-resolution 2008;8:59. melting in the context of legislative requirements for 19- Batnyam N, Gantulga A, Oh S. An Efficient validation of analytical procedures for diagnostic Classification for Single Nucleotide Polymorphism (SNP) applications. Expert Rev Mol Diagn. 2012;12(1):39-47. Dataset. In: Lee R, editor. Computer and Information

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