م د - mums.ac.ir...Amplification Refractory Mutations System-PCR 0ff2C TˇffB2E 6 0ff ff ˛ 0 ff28...

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Transcript of م د - mums.ac.ir...Amplification Refractory Mutations System-PCR 0ff2C TˇffB2E 6 0ff ff ˛ 0 ff28...

Page 1: م د - mums.ac.ir...Amplification Refractory Mutations System-PCR 0ff2C TˇffB2E 6 0ff ff ˛ 0 ff28 6 +,ff -+ I ,, V
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Page 3: م د - mums.ac.ir...Amplification Refractory Mutations System-PCR 0ff2C TˇffB2E 6 0ff ff ˛ 0 ff28 6 +,ff -+ I ,, V

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#&'(��) .............................................................................................................................................. 13 +, �- .�/�PCR .............................................................................................................................. 16

#�01 ��2/��� �)��03 ��4PCR .................................................................................................... 19 +, �- ��05 6�2� ���� ����PCR .......................................................................................................... 20

6�2� ���� ���28) +, �- ��8�� ��05PCR .......................................................................................... 28 :-� ��(���PCR ............................................................................................................................... 31 :-� ;(�<�PCR ................................................................................................................................ 33

� �)"� :*"+ ,��-� �. ���/01 ��2PCR................................................................................................................ 37

2 -1- Hot start PCR .......................................................................................................................... 37

2 -2- Touchdown PCR(Step-down PCR) ........................................................................................ 38

2 -3- PCR ��� ?(�$)(Booster PCR) .............................................................................................. 38

2 -4- PCR �(�)�,1 ............................................................................................................................ 40

2 -5- PCR #�A �( ����� �� .................................................................................................................. 41

2 -6- (RT-PCR) Reverse Transcriptase PCR ................................................................................. 42 2 -7- Inverse PCR ............................................................................................................................ 43 2 -8- (Polymerase Cycling Assembly: PCA) Assembly PCR ....................................................... 45 2 -9- Asymmetric PCR .................................................................................................................... 45 2 -10 - Ligation-mediated PCR ........................................................................................................ 45 2 -11 - Multiplex Ligation-dependent Probe Amplification (MLPA) ........................................... 46 2 -12 - Semiquantitative PCR .......................................................................................................... 48 2 -13 - Real time PCR �(PCR �� (Quantitative PCR)................................................................. 49 2 -14 - 6� 2�4 #5 #�B5�- 02C ) (Helicase-dependent amplification) ................................................... 51

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2 -15 - PCR D�2EF2�� �3��� �G(-(Methylation-specific PCR: MSP) ............................................ 51 2 -16 - Long-distance PCR ............................................................................................................... 53 2 -17 - Miniprimer PCR ................................................................................................................... 54 2 -18 - Overlap-Extension PCR(OE-PCR)...................................................................................... 54 2 -19 - PCR H���I 6� .......................................................................................................................... 56 2 -20 - Thermal Asymmetric Interlaced PCR (TAIL-PCR) ........................................................... 57 2 -21 - ��4���� J(0E 02C )cDNA ...................................................................................................... 58 2 -22 - �4���� J(0E 02C )� ����K........................................................................................................ 60 2 -23 - 02C )DNA #�01 �� J(0E ��4 ................................................................................................ 61 2 -24 - PCR �0�K� ��4�2)�L� ����2M� �5............................................................................................... 62 2 -25 - PCR ��, �0� ���0 ) �M��) ...................................................................................................... 63

05 �,�N� �M� M�� ���-�PCR ........................................................................................................... 63 2 -26 - ��� 0O�-�P� ��<QR �M�S TB2H��� ......................................................................................... 64 2 -27 - ��� ��� 02C ) ��<QR �M�S TB2H��� ......................................................................................... 65 2 -28 - PCR �M��) 02U�� ���<) �5 T40E ?� ���0 ) ��4 ..................................................................... 67 2 -29 - Inter Simple Sequence Repeat PCR: ISSR-PCR ................................................................ 68 2 -30 - V <,� 02C ) T�B2E +I ��,, .............................................................................................. 69 2 -31 - �H���) 02C )DNA ��� W��� (RAPD) .................................................................................. 72 2 -32 - XMY �G(- �2)�L� ����2M� (ASO) ............................................................................................... 73 2 -33 - V ��-�-0�4 �E�05 (HA) ...................................................................................................... 74 2 -34 - �(�ZH +(��Y �� ��,1[) #��� (SSCP) .............................................................................. 76 2 -35 - ���)��� ?��(��0O 6��H-0� M� .K ��,, (DGGE) ....................................................................... 76 2 -36 - 02C ) X5�R \-0� ](�,1 D�2E��(0N24 (MAPH) ........................................................................ 78 2 -37 - A�5 [2 ^) �5 \-_ �,P,� �E�05 (HRM) ................................................................................ 80 2 -38 - ?��� �����RNA 6� ���^�E� �5PCR ������0R ��40�(�0� �5 (RAP-PCR) ............................... 80 2 -39 - �2)�L� ����2M� .��)� D��6Y (OLA) ......................................................................................... 82

T� ��4�05�� PCR ........................................................................................................................... 83

� �)�� :*"+ /��. ��2 4/��" �5��PCR........................................................................................................... 89

+, �- �OG(- ��N5PCR ................................................................................................................... 90 +, �- ?2E�B3 +(��H� PCR ............................................................................................................. 98

+, �- ?P/ ��N5PCR ................................................................................................................. 100 ��,, ��� +, �- ��4PCR ............................................................................................................... 101

:-� ��,, ��� W`3 ��4 ��4PCR ................................................................................................. 104

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� �)+��8 : ��9:; 4/��" $<:=� >�+PCR "RT-PCR ......................................................................................... 109

:-� �F � X3 ��4PCR ............................................................................................................. 114 1 - �M��) �� �Q� ......................................................................................................................... 114 2 - �6���� .��P� ?E���� �PCR .............................................................................................. 115 3 - ��I- ���.��P� ................................................................................................................ 116 4 - �/�����02a .��P� �( .��P� ](�,1 ��I- ...................................................................... 119 :-� �F � X3 ��4RT-PCR ...................................................................................................... 121 1 - ��I- ��� �( T .��P� .��P� RT-PCR .......................................................................... 121 2 - .��P� ��I- ���PCR 6��OY .K �E�05 6� V� DY D��5 T �( ........................................... 123 3 - 6��OY .K �E�05 6� V� �/�����02a ���5 ��I- .................................................................... 125 4 - .��P� �M��) �� �)�4�N���PCR ���� ��I- .......................................................................... 126 5 - #�01 ]2M-� �� .��P� ��I- ��� ......................................................................................... 126 6 - .��P� �-�-PCR ��b��� 6� 0�25 �M� M�� D6- �5 .................................................................. 128

� �@�/A :$�B��C�.............................................................................................................................................. 131

�028�6 +, �- ��NE�P� ����� 6�0� ................................................................................................... 131 ?R� #5 c�50� �F � D�0 X3 ?b�a - �6�E ............................................................................... 133

+(��6Y �0��6�5 ............................................................................................................................... 135 .-� +'5 ��NE�P� : +, �- ��N2 0) 05 ��-0�PCR ......................................................................... 136

1 - 0�(�0� c��'� ........................................................................................................................ 136 2 - ��M�� DNA.......................................................................................................................... 138 3 - 0H�5PCR ............................................................................................................................... 138 4 - dNTP ................................................................................................................................... 139 5 - Taq ��� 6�0� .......................................................................................................................... 139

�-� +'5 ��NE�P� :��(�2,� �(0� ?b�a022U) ................................................................................... 140 :-� �4��(��6Y �� T� ��4 ......................................................................................................... 145

��(�2,� ?b�a T2b,) ���� ](0�) ....................................................................................................... 147 ���� ](0�)0�(�0� ?b�a 022U) .......................................................................................................... 151

6��H-0� M� ......................................................................................................................................... 156 e2P� 6��H-0� M� ��4 ........................................................................................................................ 158

6��OY .K �-� 6��H-0� M� ................................................................................................................. 158 ��� .K �5 6��H-0� M� Y�2�Y X(0 ........................................................................................................ 166

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AFLP Amplified Fragment Length� Polymorphism�

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ARMS-PCR Amplification Refractory Mutations� System-PCR�

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ASA Allele-Specific Amplification XMY �G(- 02C ) ASO Allele-Specific Oligonucleotide XMY �G(- �2)�L� ����2M� ASPCR Allele-Specific PCR �028�6 +, �- �� ��� 6�0� XMY �G(- BSA Bovine Serum Albumin ]2��NMY �0E�-�O CTAB Cetyltrimethylammonium bromide X2�E �0) X2���(��-05 ��2���Y dATP Deoxy Adenosine Triphosphate �0) ](6���Y �B �� ��^BH

dCTP Deoxy Cytidine Triphosphate �0) ](�2�2E �B �� ��^BH

DGGE Denaturing Gradient Gel� Electrophoresis�

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dGTP Deoxy Guanosine Triphosphate �0) ](6����O �B �� ��^BH

DMSO Di Methyl Sulfoxide �(�B �^M�E X2�� �� dNTP Deoxynucleoside Triphosphate ����� �� �� ���� �����

DOP-PCR Degenerate Oligonucleotide Primed� PCR�

PCR �0�K� ��4�2)�L� ����2M� �5

DTT Dithiothreitol �� �2).��(0) dTTP Deoxy Thymidine Triphosphates �B �� �0) ](�2�2) ��^BH

EST Expressed Sequence Tags NB105�� ��� D�25 �M��) FISH Fluorescence In Situ Hybridization #O�-� �6�E�I �� ?,E���H

FRTE Fluorescence Resonce Energy� Transfer�

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gp32 T4 bacteriophage gene 32 DK32 K�H�(0� �5T4 GSP Gene-Specific Primers DK �G(- ��40�(�0� HA Heteroduplex Analysis V ��-�-0�4 �2M��Y HMA Heteroduplex Mobility Assay V ��-�-0�4 g0P) D��6Y HRM High Resolution Melting A�5 [2 ^) �5 \-_ �,P,� �E�05 IRS-PCR Interspersed Repeat Sequence PCR �028�6 +, �- �� ��� 6�0� ��, �0� ���0 ) �M��)

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MAPH Multiple Amplifiable Probe� Hybridization�

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MLPA Multiplex Ligation-dependent Probe� Amplification�

�,1 D�2E��2M #5 #�B5�- \-0� 02C )�(�)

MS-FLAG Methylation-Specific� FluorcentAmplicon Generation�

D�2EF2�� �/����� ?,E���H �02C ) #<QR �2M�)

MSP Methylation-Specific PCR �028�6 +, �- �� ��� 6�0� D�2EF2�� �G(- OLA Oligonucleotide Ligation Assay �2)�L� ����2M� .��)� D��6Y PCR Polymerase Chain Reaction �028�6 +, �- ����� 6�0�

PEG Polyethylene Glycol ��� .� (FO ]�2)� qPCR Quantitative PCR �028�6 +, �- �� ��� 6�0� �� RACE Rapid Amplification of cDNA Ends ��4���� J(0E 02C )cDNA

RAGE Rapid Amplification of Genomic� Ends�

����K ��4���� J(0E 02C )

RAPD Randomly Amplified Polymorphic� DNA�

�H���) 02C )DNA ��� W���

RAP-PCR RNA fingerprinting by Arbitrarily� Primed PCR�

?��� ����� RNA �5 ���^�E� 6� PCR �5 ��40�(�0� ������0R

RCDA Rapid Cycle DNA Amplification 02C )DNA #�01 �� J(0E ��4

RFLP Restriction Fragment Length� Polymorphism�

��� �-�P� ��<QR �M�S TB2H���0O

RT Reverse Transcriptase h� <� 6��i(0 B��0)

RT-PCR Reverse Transcriptase PCR �028�6 +, �- �� ��� 6�0� 0) �5�h� <� 6��i(0 B�

SSCP Single Strand Conformation� Polymorphism�

[) �(�ZH +(��Y �� � �,1 #���

SSR Simple Sequence Repeats ��0 )���E �M��) ��4 STR Short tandemrepeats ��)� �H���) ��4��0 )

TdT Terminal deoxynucleotidyl� transferase�

�B �� �(���� 6�0^B��0) �2)�L� ��

Tm Temperature melting \-_ ����

TMAC Tetra Methyl Ammonium Chloride �0�)��^BH ��2���Y X2�� UDG uracil DNA glycosylase X2E���(DNA 6F(6� 2�O UV Ultraviolet Light +^,5�0H ���

VNTR PCR Variable Number of Tandem� Repeats PCR�

+, �-�028�6 �� ��� 6�0f� �M��f) ���0f ) ��f40E ?� 02U�� ���<) �5 T4

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1. Kary Mullis (1944)

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1. Cetus Corp

2. Henry Erlich

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3. Repair Replication 4. Primer

5. Perkin–Elmer 6. Human Leukocyte Antigen

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���5 #���� �2)�L� �� .T(��Y ��� 6�0� �,��� ���03 #5 �-�$�Taq ��� 6�0�� ���^�fE� �5 �� �0H�5 +, �- 0� ?^I [( 6��0) �2)�L� �� �B �� ��1 - 0�(� ��^BH3 �2M�)� �� D�2�2� # �, �f4 ?f���-�

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1. Stephen Scharf

2. Template

3. Deoxynucleoside triphosphates (dNTP)

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2. Primer 3. Annealing

4. Extension/Elongation

5. Temperature melting

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�(��� X2� ). 1. Incubation

2. Annealing Temprature

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1. Stabilizers

2. Enhancers

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1. Thermus aquaticus 2. Escherichia coli

3. Proofreading

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1. Thermococcus litoralis 2. Pyrococcus 3. Processivity

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����� 0�(�0� -� �3�0S 0O� ���N� ] �� X�� �M��) #5 �E0�E� ��� X2M� #5 �� ���^�fE� �5 D��) D�-�H� �5 #2M-� 0�(�0� �M��) 6�2 �(3 ����� #5 �2)�L� ��′3 �0f �f3�0S �� �-� 0�(�0� � . #f ,(� �f5

�M��f) �/����� 02C ) m��5 ��� ���� #2M-� ��40�(�0� �5 �I�) X5�R �����i�4 ����� ��40�(�0� - W�4�/�����02a �A��P� W`3 �� ��� .?M�3 ](� �� #�NM� T(�f�Y 6� �(�N� ��f4 ?f2M�<H �����

�Q� �2P�)′3 ����� �) �0 ���^�E� �6�L� ��-�O�′3 ]22<) ��,, ��� �^3. #M�M #5 .-� #�H�( 02C ) .��P� .�$��� �S �� �O��MY .���3� :-� ](� .� �� 0f2C ) # ?E� ��

��8�� DY �� �-��� �4��� .�O��MY �� ���) .��)� ���� �5 ��� �3�0S ��40�(�0� 6� ���^�E� �5 � 0f2C ) c�f�'� -� � (�2H �6�E��I ��05 kM�� ��2B5 ]a-� D�-�H� #�2E- #5 ]2,&�4 - �-�^��

��� .0�, .

2-6- (RT-PCR) Reverse Transcriptase PCR�

6� q�� ](�PCR 02C ) �( x�0'�E� ��(�E�,� ��b,� #5RNA [) #��� #f���� 6� �� �f( .��fE ��f4 �� ��0R ���^�E� ���� ?H�5 �02O . # �(�8�Y 6�DNA ��� 6�0� 6� ���^�fE� #5 ���RRNA �f�M� D��f,� #f5

��� #5 �0�(� #�30� ���5PCR ?E� ��� #H�t� . h�f <� 6��i(0 V��0) T(��Y 6� #�30� ](� ��1 X(�N) ��05RNA ) �G(- #5mRNA ( #5cDNA ���^�E��� ��� . ](� ViEcDNA #�2fE- #5PCR

�� 02C ) ��0O .�0� ����EF5��2� h-0(- 6� h� <� 6��i(0 V��0) T(��Y2 �� ?E� #5 �(Y . �05��f �� T ��� #5 D��5 h�B3 X2M� #5 T(��Y ](� ���5 . ]f(� h�f�2H��0) h��0) �0� �5 l �5 �M-:-�

?H�( ��N5 . �0� �5 ](�DNA ��� 6�0� ���03 #5 �-�$� �� �2M�) ����� �f,�,� D�( ��Z3 �� - �, #'B� ?2M�<H ����05h� <� �� ���5 . ���� �� ]2,&�472 #fI�� �f� f���) �-� 6� �RNA �f�M�

DNA �6�B5 .�2E� [2�E� .� 2�O ]�2)� eE�) �,�,� W`3 �53 6� T(��Y ](� �DNA ��f� #���fE�� #���� �� 02C ) - ��0 ���^�E� �4� . q�� ](�PCR k2'f) ��05 h-0f(- ��f4RNA 0f2b� ���

HIV ?fE� ��f� ���^�fE� �f2� DK D�f25 �,) D-05 #<M�Q� ?I �2� - �5-0 2� �t D���� �5�(6�� - �0(6DNA �M��) ���Y ?E� #5RNA �� �^3 �� ��/� �f, . f��O ]2f2<) ��fb,� #f5 ]2f,&�4 - #

h-0(- �� �(-��� ?2E�B3 0f2C ) ��05 :-� ](� 6� �K�M�(0� �5� (�� - �E�,�RNA ��-6�fN(� ���^�E��� ��� . #f���� �:-� ]f(� 6� ���^�fE� ��/� +M�1mRNA ?fE� - y�QfE �f5 ��f �0f(6

1. Reverse Transcriptase

2. Avian myeloblastomavirus (AMV) 3. Aceticacid ethylene glycol

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43 PCR 7� � � :9� :+� �� �$��;& ��6

?b�a ]2(�� ��4mRNA +,f �- #f5 ?2E�B3 �5 ��0�4 n�)� ���� �� DY T ����(�� - 0b� �������N(�X � e2P� #�(�2E� 022U) - �46�L� �� ���5.

��2-3: ��� :A� 8�RT-PCR . �� ��� T&� �*cDNA ���� ��M�� 6���; >!PCR �* Y�)� �� *��.

2-7- Inverse PCR

W�4 :-� ](� �� #<QR 02C ) 6� ��DNA #��(�� �M��) ���� �� ��FS� r24 # ?E� DY ��f4?E� ��� �(�E�,� ����� �M��) �( V�� E [( e$H - ?B2� ��I�� . ��0f5 :-� ]f(� 6� A�f�<�

�M��) �(�E�,� �-�8� ��41 ���^�fE� ��f� n�PM� ����K ��<QR D���02� �� �f� ��f� . ��fb,� ](�f5 ��f<QR ���f� #f5 ��f� .�f�)� VifE - ��� TZ4 ;E�,� ��,, �-�P� T(��Y eE�) ��M� �M��)

�� n�^)� #�/�3 �M��) # ��H� ��� #���,� ��4 �f� ��f8(� #���,��� �M��) ����� -� 04 �� �� �f, . ��� #���,� �M��) ��05 ;E�,� 0�(�0� �3�0S �5 .�3�� D��) �� #���,��� �3������� 02C ) . :-�

PCR ?B(6 �� ���(6 ��4�05�� h� <� �M��f) �(�fE�,� - 0f2C ) �f,��� �M�f M�� �E�,� ��f4 1. Flanking sequences

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g0P�� 0/�,� W�0S�1 �� ���- ���K �� �H���) ��S #5 # ����K ���� ��f�(�I �(�E�,� - ��f4h-0(- n�PM� ���� �4 .�� X(_ y0� #5 :-� ](� ��8�� X3�0� ���5:

1. DNA �B4Y #5 W�4 �f,1 0� 1�f ��f<QR #f5 6�L� ��-�f�� T(��Y �5 TZ4 #�2E- #5 ��:05 �6�5��2 �� ����.

2. ?b�a �� ��<QR ���� #5 ��� .��)� �� �� ]2(�� ��4 08,� # �4��� ��f� \�1��f1 �02�5 X � ���5-� �)�^BH . #�30� ](�DNA ��8(� ��$�3 �� �, .

3. DNA �-�P� T(��Y �5 ��$�3 W�4 ��,, � #���,��� TZ4 �� ��0O . ;fI�� �f,(�0H ](� ��8(� - ��$�3 �M��) :05DNA ��� #���,� ����� -� �5 �Q� �� ��� �M��f) ]f(� #f

�� ���) �,(�0H ��05 �PCR �02O ��0R ���^�E� ����. 4. ��f��� -� �� D�f, � #f ��-�� ��� #���,� �M��) X� � ��40�(�0� 6� ���^�E� �5 02C ) �,(�0H

DNA ��8�� ?E� #�H0O ��0R W�4 �� ���.

��2 -4: ����/ �& ) �� ����*��5� :&% ? ��! #5� ����* ��� >�2 �� ����/ F& f��D� �* >�2 �� � H.

1. Transposable elements

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45 PCR 7� � � :9� :+� �� �$��;& ��6

2-8- Assembly PCR )Polymerase Cycling Assembly: PCA(

��fM� M�� ��,fE ��05 �4��(��6Y :-� [(DNA ���f5 � ?fE� . .�f��� �f5PCR �-��M��) �5 X(�S ��4�2)�L� ����2M� 6� �<N,� �(�4 ���,f�� ��,E ����� �4�)� D���i�4 ��<QR #

�M��) X(�S ��4DNA �� ��8�� �02O .����2M�?I �� �4�2)�L� �f��Y - V,E ��4 V,fE X f� #f5 ��0R \-�,�� �� ��<QR ;2)0) D���i�4 ��<QR - ��02OPCR ]22<) �� �� fI�� #f5 #f �,, DY ;

X(�S - �(�� .��P� �5�'��� ��S #5DNA �2M�) �� �� �,, .

2-9- Asymmetric PCR�

�2M�) :-� ](� �02O�� #5 6� W�4DNA [) #�f�� �f� �� �f��5 . :-� ]f(� �� ?f$2$3 �� #��� -� 6� � ( e$HDNA 02C ) �f� ��f�. �M��f) �� :-� ]f(� �5�f( #fO�-� - �6�fE \-0f� �f5

���^�E� �� ��� ?fE� 6�f2� Xf� � #�f�� -� 6� #��� [( e$H 02C ) # . :-�PCR .�f�<� XfC���8�� �� ��� c�f�'� ���- �0�f25 D��f2� �f5 W�f4 #�f�� #f5 c�f50� 0�(�0� ���PCR �f� ��0fO

0�(�0� -� ?NB� # �(�I �)1 #550 �)1 #5100 ?E� . �0f�(�0� ��f�)� 6� Vf� �f, 02C ) 0S�� #5 ���� �0f�� ���f$� # #f�01 ���f<) ?fE� �6A � ��f4PCR ��f� #f�H0O 0fb� �� 0�f25 . 6� Vf�

20 ���� T1 X5�R �3 #5 .-� 0�(�0� ?b�a # .-� #�01 �� �f5 �f�M� 6� #�f�� [f( ef$H ��E�02C ) �-� 0�(�0� �� ��� . D�(�� 6� V�PCR #'B� �,1 �DNA #��� -� �� ���f(6 ���<) -DNA

[) #��� ��(� 0b� ���� DK 6��� �4��� ��8 . #f ?E� #�H0O ���/ :-� ](� �� ��(�I 022U)Linear-After-The-Exponential-PCR (LATE-PCR) ������ �� ��f� . 0f�(�0� :-� ]f(� ��

���^�E� 0�(� 0�(�0� 6� 0)A�5 \-_ ���� �5 �-�P� �� ��� ��� �^3 +, �- �(��� �).

2-10- Ligation-mediated PCR�

6� :-� ](� �����^�E� �4������Y �( �40 ,2M �� ��f<QR #f ��0fO ��f2)�L� ����2M� [f1� DNA ��<QR #5 - �,�B4DNA X��� W�4 �� ���� . ��40�(�0�PCR �M��f) #f5 # ��f4 0f ,2MX��� �f� 0f5 ���f� ���^�fE� ���f� W�f4 �M��f) 0f2C ) �� ��0fR �f� �f�02O . �f�M� #�f�� �f8,(� ��

[) #��� ���0) 6� �'5 # ?E�0�25 �M- k'� DY W� +H��0) k'��� ?E�. ��f8�� ��05](� :-� ���5� 5# #�2E- [( 0�(�0� ��� #5 �/�����Extension Primer .�f�P� - #���E X� � #���

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46 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

#��� -� 0 ,2M [( #5 X��� ���� ��� . �0f�(� - 0f ,2M j�f�'� �f ( # 0�(�0� -� #�2E- #5 ViE X�� ?E� W��0) j��'�PCR �� ��8�� �02O.

�M��) ��05 :-� ](� �5�(DNA����K ��6 ��R �1 ?��� - �����DNA2 �f� �� #5 �-� .:-�

��,(�0H ](� �5 #5����� ��f� 02C ) ��<QR �M�S TB2H���)AFLP(3 �f�2') ��f<QR #f ?fE��� �2M�) �� ���K �, .

2-11- Multiplex Ligation-dependent Probe Amplification (MLPA)�

�0�(�0� ?^I [( 6� ���^�E� �5 :-� ](� ���� D��) q�� �,1DNA �0f 02C ) �� l��'� � .#O� -� h�E� 05 :-� ](� �6�E ViE - ��M� �5PCR ��8�� �� Ff/� �5 - �02O DY �-� #f �)�3

h�B3 #�H0O ���/ ���E - 0)0u�� �0) 0)?E� ���. :-�MLPA �� ���) 6� �NB� �� �5�(6�� [( �50 02C ) l��'� .��P�PCR �f4� #s���

�S �� +, �- [( eE�) ���4 # 24 �)48 ��� �2M�) ?��E ���. �M��f) �f,1 :-� ](� ��) �f)50 �M��)DNA (�� 02C ) 0�(�0� ?^I [( 6� ���^�E� �5 [f2 ^) ?(�-�fP� 6� ](�05�f,5 ����� �0(`f�

�� # Multiplex PCR � ��I-�� �02O��I ��� �, .�8�Y 6� 0f2C ) #f82�� �A�f�P� ]f(� #f \-0� �M��) ��� #� - ��� X��� ��4DNA e$H ��,�B4 �E�05 ����50 �f)250 6� �0O���f�DNA

�� �H� - �6A +, �- [( ��05 ����K ���5. +, �-MLPA +'5 X��� �� 0(6 ��4 ���5: #��� -� D�� 6�5DNA #O� -� ���M� �6�E\-0� �M��) �5 �4 ��4 \-0� .��)� �X� � W�4 ��f4

02C )PCR \-0� ��46��H-0� M� - ��� X��� ��F2i 4 Vf� ?fE� ]f �� - �A��P� DY 6��02O ���/ �A��P� �E�05.

�3�0S ��2)�L� ����2M� \-0� -� +(��6Y ���� �M��) 04 ��05 �f� ��f� Xf� � #f �3��f��,�B4 T4 [(��� - #(�B�4 .� �<5#O� -� 6 �6�E �5 \-0�DNA \-0f� .�f�)� ���M� ���f/ �f4

�� �02O . +, �- ����Y \-0� .��)� 6� �<5 e$HPCR ?E� . #fO� -� D�f1 �6�fE ��0f5 .�f�)� -�?E� ��-0t \-0� 02C ) �f�� X��� W�4 #5 # �(�4�2)�L� ����2M� �� Xf�� \-0f� - ���f�

��� +, �- �� ��6�EPCR 02C ) ��� - ����DY W`3 ��05 ��B� #5 6�2� ](�05�,5 ?fB2� �4 . �M��f) 1. Genome walking

2. DNA footprinting

3. Amplified Fragment Length Polymorphism

4. Capillary Electrophoresis

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47 PCR 7� � � :9� :+� �� �$��;& ��6

�(0N24 ����� ]2f5 �M�S \-0� 04 �� ��I��55 �f)80 ���� �f2)�L� �� . ��f2)�L� ����2M� \-0f�o1 )LOP(1 ?^I �M��) [( ���P� ?E� 0�4�)� # ����� � ����� �� 0�(�0� #55 ��� �f� �f��5.

?E�� ��2)�L� ����2M� \-0�)ROP(2 # �� 0)�,�5 ?f^I �M��f) ���P� ���5 ����f� 0f�(�0� #f5� ?�E �� # ?E� �-�3 ��� #5 0�(� �M��) [( - ?E� #�H0O ��0R DYStuffer DY .�fS #f ����E� �-�^�� \-0� 04 ��? .[( e$H #f�4 02C ) ��05 0�(�0� ?^I \-0f� ���^�fE� �f4 �f� ��f� -

� ��8(� �� �-�^�� .�S �5 ��� �2M�) �A��P�� �, . #���8� +, �- 04 �� .�S �5 �A��P� 6�64 �)500 �2M�) �� �2)�L� �� �� #�2fE- #5 # �,

��I T4 6� ��F2i 6��H-0� M� �� ���� ..0�, �5 #B(�$� #�2E- #5 r(��� ��4 ��f<QR �f5 �f( - TM�fE�E�05 ����� .0�, �� ���� . ]25 [2� ?NB�7/0 �)3/1 6� 0)A�f5 ?NB� ��<2NS3/1 D�f� ��f,4�

��� �M��) +(��H� 6� 0�� ?NB� - D��6Y �7/0 D�� ��,4� ?E� W�4 D��� ?E� 6�.

�\\�2 -5: 4$$�� [L :$$A� 8�MLPA . ( $$G/� :$$H �$$8&*% �* f�$$H ����$$/ #$$+8� � H�$$�/�g�� �M��� ( $$G/�

! ��E8/ � 6��) M�� ���� ! h7 � 4)�� � H��/�g�� �M���PCR U��\ �� *��-.

1. Left probe oligonucleotide 2. Right probe oligonucleotide

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48 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

2-12- Semiquantitative PCR�

#���� [( �� ��I�� [2L� �� �2E� �NB� ���$� ��05 �,2�') :-� ](� �� ���5 . #f���� �f�R-RNA ?E�cDNA :-� �5RT-PCR �� ?E� #5 �(Y ..0�, ViE ����� ��4) 0 ��f� D��f,� #f5 #

�� ���^�E� ���� (�� 02C ) ���� . �f� ���^�E� r(�� ��S #5 # �(�40 ��� ���f�Apo A1 ]2f� � -B �,�B4 .��I 6��H-0� M� #�2E- #5 ��� 02C ) .��P� �f� ��f� . �f�� 6� �f<5 ��f2�Y �f5 6��fOY .K

�(��05 ��(�2)�V � ����05 ���8�� .K 6 �� #NfE�P� 0����2fB�� eE�) ���� �M��1 �6���� - �02O �� ��0O .#O�-� .���3� �:-� ](� .� �� �6�E�2M�) \��Q��� r(��� # ?E� �G(- 02a �f� �f, .

\-0� 6� ���^�E� �5 �OG(- .0�, ��4#O�-� ��05 �G(- ��2� �6�E��8�� �� �02O.

��2 -6: ���Semiquantitative PCR . : �&* B� �& ) �� ���! ��� T&�DNA �<$A ���$B� >$A& B� � 4$)� ��Y�

(��� ! (�G5� � HU��\ ��2�* �� *��-.

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49 PCR 7� � � :9� :+� �� �$��;& ��6

2-13- Real time PCR ��PCR ��� )Quantitative PCR(

:-�Real time PCR �(PCR �� )qPCR(1 :-� 6� �0�(� z5�Q)PCR ]2f2<) ��b,� #5?���-� ���<) �� ��4+, �- �S �� [2L� �� �2E� PCR ?E� . ](�05�f,5qPCR ]2f2<) ��0f5

���$�DNA �(cDNA ���^�E��� ��� #���� �� 0t�3 ?���-� �( DK ���<) �) ��4 lf�'� ]2f2<) �� �, .?P/ 0b� 6� :-� ](�2 ���f(��6Y �� - ���� ��0fR �QE ](0)A�5 �� ��f4 k2'f) �,2M�f5

<2E- �05�� ?E� #�H�( � .](� ��(��� #��I 6� :-� �O��fMY 0fQ� +4� �#82�� .��3 �� ?�0E ��-�,H �02O�� 5 �� ?M�E - �� �,��5. [f( �f�2') T�B2E ](� �� �fS �� ?��fE���H ���f�

2E04 �A��P� D��2� �5 ;E�,�� +, �-��6Y X �� ��� - ��f� )� efE�) DY ?��fE���H D��2� �� ?Nu - �(�E�,� D��2� ;2)0) ](�5 - ��0ODNA �� ��f<5 #�01 �) #�01 [( �S ��� 02C )

�� �6���� D��) �0 �02O. E� �5�(�� X5�R #�0104 �� .��P� D��2� ](�05�,5?. ?E� q�� -� A��<� :-� ](� �� V��E���H �5�(�� T�B2E :�� x�� Xf�� ����f� ) #fM� 0�,(� ( -

\-0� ��4D�� ����4��H-���H �5. x�� X���� ����� 02b� SYBR Green �-0 -��f�H #f �,�fB4 �(�f4 �� V��E���Hz(0S 6� [1� ��2� #5 .��)� DNA #��� -� ��+(��H� 02��1��S #5 �� �f,4�.

��� ](� x�� .�S \`I �5 �4 498 ��� ��0������ 522 �0������ �� JS�E �� �,, . ���f$� +(��fH� �f5 .��P�PCR �� X��� �0�25 ��� ��� +(��fH� V��fE���H D��f2� - �f� ��f5�( D��f2� ](�05�f,5

V��E���H ���$� �5 ;E�,�� ��� 0�,�DNA #���-� ��?E� +, �- ��. #f� � ]f(� #f5 �f(�5 #f�NM�x�� X���� # ?��� #I�) ����� �0(6 ����� �� �OG(- �� ���) �f/����� 02a �A��P� #5 �,

- ���� X��� 0�(�0� ��40�(�� - �f,, ��-Y0f5 ��f/� ?b�a 6� 0�25 �� r(��� . ��f<M�Q� lf��'����� D�� ��� #,25 e(�0� 6� ���^�E� - 0�(�0� z2R� \�'��� # PCR ?2f/����� ��� ?E� ] ��

�4� +4� �� . fE +,f �- q-0� ��05 A�5 ���� 6� ���^�E� ��,)Hot-start PCR ( 0f2C ) 0fQ� �f2�+4� �� �/����� 02a �� �4�.

�� :-� �� # �0�(� �5�(�� T�B2EPCR ���^�E� �� �� ��� \-0� #fO� -� �Gf(- ��4 �6�fE D�� ����� -� �5 ���V,E���H ��� [( X��� �-0 -���H q �� �6 � �����5 )Reporter ( �f�� [f( -

:���� ��,, � �������3 )Quencher( �� �f���� ��0fR �f (��� �M�f M�� #�/�H �� ��R- - �,��5��� ?Nu ����E� �� �5�)6�5 6� ��f� JS�E ��� �(��I 6� V� ��� ���Reporter efE�)Quencher

1. Quantitative PCR

2. Accuracy

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50 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

�6���� X5�R V��E���H ���/ #5 ����E� eE�) - ?B2� \`I X5�R �02O ?fE� . WF�0f5 �f5-0�E�05 ](0O0N(�E�� .��P� �M��) �/����� k2') h� �� �,, .r(�� \-0� ](0) ��4 ���^�fE�

\-0� ��� ��f4 �f( ��2M-��f24TaqMan �M�f M�� ��f5-0� �Beacon -FRTE ) �K0f�� .�f$���?,E���H V���-6�1 (�,�B4 . \-0�Taq Man DY h�E� # ?2M�<H �6�fL� ��-�O�DNA �f�� 6�0f�

Taq ?E� � \-0f� [(30-20 � ��f��� �� #f ?fE� ��f2)�L� ��5 - V��fE���H �f�� [f( #f5 � ����� ��3 :���� X��� [( #5 ��,, ?E� X��� .�,1 �-�3 \-0� �f<5 6�f5 � ��f��� 6�3 6� �f (

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�� V��E���H ��0O . +(��H�DNA #O�-� �5 ;E�,�� #�01 04 �� �6�E\-0� �f2M���� #f ?fE� �f4?E� ��� 0�,� V��E���H +(��H� �5 ;E�,�� .\-0� ](� �,104 ��2&2� �4 0)D�0O - ?f�2R 6� 0f) Xf����

x�� ��,�B4 ����� \-0� ](� 6� ���^�E� �4 ��� �(�E�,� TB2H��� D�2E�)�� - �4 �� ] �� �� �4 �6�E.

��2-7: :�A�) �� �* Y�)� � H ��� �* i���� 4�)���LReal time PCR.

1. Fluorescenceresonance energy transfer

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51 PCR 7� � � :9� :+� �� �$��;& ��6

2-14- 5�:�%2 . �R.�" ��9:;)Helicase-dependent amplification(

#��� -� D�0 ��I ��05 [2, ) ](� ��DNA 6�f 2�4 T(�f�Y 6� ���0f3 ��fI #f5 �1 ���^�fE� �� ���. �M��<� :-� �,��� :-� ](�PCR �� ��8�� #�f�� D�f� 6�f5 X3�0� �S �� ��� ��0O ��f4

X(�S - 0�(�0� .��)� �6�E�� ?5�u +, �- ���� ���5.

2-15- PCR ����- LS�"����<��� (Methylation-specific PCR:MSP)

](� :-�PCR �E�05 ��b,� #5 ��f<QR �� D�2EF2�� �3��� CpG ��DNA - ���f5 ���f�K .�fE �� ��f5 ]2M-�1996 efE�)StephenBaylin -Jim Herman ���f��� � f��� �� f��� ��

�,���E :-� [( #5 ViE - �� q��5� �,2 ��4 D�I D�2fEF2�� ��4��M� �(�E�,� ��b,� #5DNA ](6��2E 0(��I �� - ]2���O2 )CpG ( ��DNA �� X(�N) ����K. ?f^I -� :-� ]f(� ��8�� ��05

�3��� #5 .��)� ��05 0�(�0�CpG �3�0S�� ��� . Xf��� #�2�� �3��� #5 # 0�(�0� #���8� ]2M-��� ��� �-�34 X� � 6�5CpG �� ���5 . Xf��� #f�2�� 02a �3��� #5 # ?E� �0�(�0� �-� #���8� �� ��� .�� ]2�2) �-�3 ](6��2E 6�5 ��I #5 #���8� ](� ���5 . �f40�(�0� ](� �3�0S �� ��S #5

#��O #5 �� ���/ �� � ����� �� ]2�2) - ](6��2E 6�5 # �02O3 ��f8�� ��N�f�� .��)� �) ��02�5 ��0R�02�� .�M��) �5 0�(�0� #���8� -� ?E� �6A �2�� 02a - #�2�� ��� #���,� ��4 ���f� D�f�6Y # . 0f�(�0�

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���� #�2�� 02a.

+, �- ��8�� ��05PCR �3��� �E�05 -CpG ���5� �� �DNA T(�fE �f5 ��f�2) ?fP) W�f4�5 ��0R ?2^M�E �� X(�fN) X2fE���( #f5 �� #�2�� 02a ](6��2E ��46�5 # �02O �f� Xf� � #f �f,

�,(�0H �� ](6���YPCR ?E� . +,f �- �f<5 #f�30� ��PCR ���f/ �f� �0f2O. �f�� 6�0f� ���f�� - X2E���( D����� #5 ���R �(�5 :-� ](� �� ���^�E�5 2�����5 ](6��2E X . ��,fE #5�f� #f�30� ](�

#���DNA �-� 6�RNA ?E� h� <� 6��i(0 B��0) eE�) . ��N�f�� 0f2C ) 6� �02O��I ��b,� #5[( ?E� �6A ��� 6�0� Hot start ��� ���^�E� A�5 ��NB� ���� �5 ��0�4 .��� 6�0� ��f4 �0� �f5 �f �Y �f4

XC�Vent -Pfu 02C ) �� X2E���( �-�3 ���M� �,�B4 A�5 ?P/ ����� # ��� �,, . 1. DNA Helicase

2. CpG Islands

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c��'� # ��� #I�) �(�5 ]2,&�4 ��4PCR X2E���( ���P�DNA ���^�fE� �f(�N� 6F(6� 2�O .��P� ](� �0(6 ���� �4DNA �f� �2M�) X2E���( �-�3 ���M� - #�B � �� X2E���( �-�3 efE�) �

�5 ;(0') �� ?2^M�E �� �,, . �-� 02C ) -� ?(�� ��DNA ��8�� �� ��� : #f5 .-� 0f�(�0� #f���8�DNA ](6��2fE �����

X��� �� ��� ) ��f� #f�2�� ](6��2fE �f5 0~�,�� ( #f5 �-� 0f�(�0� #f���8� -DNA X2fE���( �����X��� �� ��� )#�2�� 02a ](6��2E �5 0~�,��.(

��2 -8: ����/ �& ) �� ��b � >���� � H >��������/ ���j/ �� �* Y�)� ! �! ! >���� � H ��E8/ � :&�) 4�Y��)PCR.

PCR �� .K #(�� 05 �^2 :-� [( D�2EF2�� �3��� �G(- �f��5. :-� �0f2�� �f2� �f� ��f4

#(�� ��� ���`O ���DY 6� ���^�E� �5 # �4�� D��) � �2� �� D�2EF2�� D��2��0 �5�(6�. �5 �� :-� ]2M-�\-0� 6� ���^�E� �2M-��24 ?,E���H ��4 ��,, ?fH0O ��f8�� . #f :-� ]f(� ��

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��� 6� ��<5 :-� �� #M� 0�,(� ?,E���H ��4�,��� SYBER Green I \-0f� ��fI #f5 ��f4 ���^�E� ?,E���H�� ��� .��� ](� #5 �4DNA #��� -� �� ��8(� .�,�2E - ��� X��� �� �,, .

��E :-�MS-FLAG1 ?,E���H .�,�2E # ?E�z(0S 6� ��40�(�0� ]�B �MSP #�2fE- #5

�-�$� 6�L� ��-��� ���03 #5PspGI �f� ��8(� ��0fO . Xf��3 �� [f( �-�f3 �f40�(�0� ��fH-���H 1. Methylation-Specific FluorcentAmplicon Generation

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T(��Y �(�E�,� D� � #�2E- #5 # �,�B4PspG1 ��I 0�(� ( 6� �f40�(�0� .�f�)� �f5 # �02O �(�fE�,� �M��f) :0f5 6� �f<5

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c�f�'� ���f/ #f5 :-� ](� �� # ��f� W`f3 T(��Y ](� .�,2�0)

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Long-distance PCR�

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�G(- c��'� A��<� �� 6� ����� 6�0� ?f2�5�R �f5 ��f4 Proofreading

��� . T(��Yklon taq #'B� 6� �� DNA ��� 6�0� ?E� c�f�'� ���f/ #f5 :-� ](� �� # #51 �5 ��� 6�0� 02b� �$2R�pfu �� ���^�E� ��0O . ?�BRN ��f� W`f3 T(��Y ](� .�,2�0) ′5 ?E� �6�L� �� -�O�.

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?NB� �5180 #5?2M�<H �R�H -

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54 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

2-17- Miniprimer PCR�

q�� [( 6� :-� ](� �� ��� 6�0� 5 ���03 #5 �-�$� �(�I # ���)S-Tbr ( ���^�fE� �f� ��f� #f ���R [1� ��40�(�0� #5 ?E�9 �( 10��2)�L� ��1 ��� X��� .�M�3 �� # Taq ��f40�(�0� #5

X��� �0�O��5�� ��� �-�3 # 20 ����� .�S �2)�L� �� . 6� ���^�E� �5 ](�05�,5S-Tbr ��f40�(�0� � �� �0� 1� �� D��) �0 �3�0S .W�4 :-� ](� �02OX��� �3��� ����� [f1� ��f40�(�0� #f5

eE�)PCR �� ] �� �� �M��) 02C ) ��05 - �6�E ?~�^3 ��4 ���DNA ���K �,��� 16S rRNA -18S rRNA ���^�E��� ���.

2-18- Overlap-Extension PCR(OE-PCR)�

���� :-� ](�PCR �� ��f� �f5 # ���5 ��f4Splicing by overlap extension -Splicing

by overhang extension (SOE) PCR #���,� �2��� ��� . ���,fE ��f40���2M� 6� :-� ](� �� �M��) 6� �/�� c�$� #5 �-�- ��05DNA ���^�E��� ��f� ?fI [f2��K �fE�,� �� A�f�<� #

�M��ff) �� �ff/�� c�ff$� �� +ffI D�-�ffH�DNA �0�(�ff ( #ff5 �ff�K ��ff<QR .�ff�)� �ff( n�ffPM� � D�0 #H�t� (�I #<QR [(�2 W`3 �( #<QR [(3 D�� - 0b����� �M��) �� �6�E ��<QRDNA

�� �� #5 �-�. 0f�(�0� ?^I [( j�� #23�� [( �� D�2E�)�� ��8(� - W�4 �M��) 6� ���BR 022U) ��b,� #5

X� � # ?E� 6�2� �I��� ��4���� �M��) W�4 �� �,��5)A -D .( 0f�(�0� ?f^I [f( ]2,&�4 � ��4���� �5 ��-�� ���^�E� X��� ��� )B -C .( 0fb� ���� D�2E�)�� ���P� ����� ��40�(�0� ](�

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A -D �� ��8�� �02O. .�f�P� - ��f� Xf��� 0�(� ( #5 �-� #�30� �� �A��P� X� � ��4���� �(��AD �2M�) �� �� �,, .?E� #�H�( D�2E�)�� ����� �M��) �-�3 #<QR ](� .�M��) ��f4 ��-�-0)�,�5 �� ���) �f,�5 j�f� ��f40�(�0� 6� ���^�E� �5 �, 0f2b� ��4�f2)�L� ����2M�IDT Ultramer™

�E���� #�� .D�2E�)�� ��8(� ��b,� #5 ��4 �f���� ��f40�(�0� ��H`3B -C #f23�� W0fS -� ���� �3�0S W`3 ��05 0b� ���� ?�BR ](� �-�- 6� �) ���� ��<QR #5AB -CD .-� #f�30� �� #

1. Smalligos

2. Insertion

3. Deletion

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PCR �2M�) �� ��f� �02O��I ���� . �M��f) ��f4 #�2fE- #f5 #f ��f<QR ]f(� ��f4���� �� Xf� � ��40�(�0�B -C ��8(� �� �-� #�30� �S �� �����R ����PCR ��f<QR �5AB #f5 ��CD Xf���

�(�� .��P� ?(�� �� -�,, AD #���E 0b� ���� #23�� W`3 �5 �� ���.

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����/ � H����/ *��� > ���AH #+8� �� � ��)�!? S�12 .( ��$�� �*PCR O $� � �$� *�$� (�� ��$�� >$

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2-19- PCR �� 5��1

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#(-� -� 02C ) 02B� ](�1 ��2����� ��� .�i ��4 �� �R�5 �QE #5 X��� ��� ��8(� #f �M�f3 �� �f,��� DNA �� 6�5 .��P� 6�H #5 D�2E���)��� #�30� 6� �<5 �(���5� ��0O . ?f���-� ](�f,1 # ,(� 6� �<5

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.� M�� 6� ���� [( �QE #5 X��� ��4�� ��� . �f3�0S �� �OG(- ](� 6�DNA microarray ?E� ��� ���^�E�.

��2-12: #I���PCR �� ' � L.

2-20- Thermal Asymmetric Interlaced PCR (TAIL-PCR)�

:-�TAIL-PCR #���,��� ��2M��) �6�E��I ��05 �� �� #5 �� �-� �M��f) [f( #f5 X��� # ��� #���,� ���. �M��) ]�H�( ��b,� #5 :-� ](� ��4 D�f � �(�fE�,� ?fI �f2� - DK �f�2b,) ��f4

1. Interfacial amplification

2. Surface amplification

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02C ) ��-�� ��40�(�0� 6� �� ����.

2-21- ��2���-� >��� ��9:;cDNA1

?B(6 �� ?f���-� [f( Xf�� �M��f) D��-Y ?E� #5 ��b,� #5 :-� ](� 6� �M� M�� �E�,�RNA ���^�E� ���� ��I- .��E [( �� # �� ��� . [f( :-� ]f(� .�f�P� cDNA #f ?fE�

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��2-13: 5'RACE.

1. Terminal deoxynucleotidyl transferase(TdT)

2. Universal primer (UP)

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2-22- ���-T ��2���-� >��� ��9:;1

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2-23- ��9:;DNA ��8 +� ��2>���1

J(0E 6� � ( ](0):-� ��4PCR ��f� #5 �/��'� ����E� #5 6�2� # ?E�Light Cycler -#M�M ���� ���0f3 J(0fE .�$��� ?I j��'� ��4 . :-� ��PCR ���f(6 ��NfB� D�f�6 �M�f�<�

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2-24- PCR L�-T� ��2 �;�U%��-�V��� �.1

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2. Fluorescence In Situ Hybridization (FISH) 3. Used PCR-Based Techniques

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2. Restriction Fragment Length Polymorphism(RFLP)

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��,�)��R �� 2, ) 6� � ( :-� ](� J(0fE - ���EPCR k'f� ��0f5 +fI D�0f ��f4#Q$� ��� ��� S TB2H���#H�t� - W`3 - 0O�-�P� ��<QR �M� ��4 �f� [1� ��2)�L� �� #f �f��5

1. Amplification Refractory Mutations System: ARMS-PCR

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�� ����� .��)� ��� X2M� #5 ��0O′3 T(�f�Y efE�) �6�fE�,���4 ���M� �M��) #5 0�(�0� �f�� 6�0f� ��� ���/ � �2M�) .��P� ](�05�,5 - �02O�� ��� .I �0�M�50a ��05+ 0�(�0� e$H j�� ��4 #�H�( +IARMS ) .0f�, 0�(�0� -� - g0�� 0�(�0� [( X��� 0�(� 0�(�0� #E �5 ��0�4 ( ��-0ft

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0S - T2b,) �:-� ](� h�B3 - X � ?�BR ��40�(�0� �2P/ �3�ARMS ?f2M�<H �E�05 - �� ��Y �,, X�� ��E�� #5 �) ���5.

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?E� ���5 ���(6-0�4 JR�- �� - #���� ��. 6� #f ?E� ](� T� #� �DNA �f�� 6�0f� #f � �fR�H�2P�) ?2/�� �6�L� ��-�O� �O�,, ′3 #5 ′5 �?E� ��f� ���^�E� T(�f�Y [f( 6� ���^�fE� �0f(6

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1. Allele-Specific PCR (ASPCR)

2. Allele-Specific Amplification (ASA)

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#��I 6� �0�(� ���E� �5 :-� ](� MAMA-PCR lf^'� Amplificatin Multation Assay Mismatch - COP-PCR l^'� Competitive Oligonucleotide Primming #���,� �2��� ���.

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�� ��& / (��� >[1= � � �� ���� 45\PCR 4)�.

1. Tetra-ARMS PCR

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2. Heteroduplex Mobility Assay (HMA)

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�(�ZH +(��Y - ?b�a �� ��022U) ���5 #���� ��I- �IDNA #f5 ?NfB� #f�H�( +IDNA ��8(� �)-�^�� ���5 �<2NS �� �, .

1. Single Strand Conformation Polymorphism (SSCP)

2. Denaturing Gradient Gel Electrophoresis

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1. Wildtype

2. Temprature Gradient Gel Electrophoresis

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1. Multiple Amplifiable Probe Hybridization (MAPH)

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2. RNA fingerprinting by Arbitrarily Primed PCR(RAP-PCR)

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1. Oligonucleotide Ligation Assay

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2. Drosophila melanogaster

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4. Saccharomyces cerevisiae

5. Candida albicans

6. Plasmodium falciparum

7. Leishmania major

8. Trypanosoma cruzi

9. Trypanosoma brucei

10. Schistosoma mansoni

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�Tm = 4 × (C + G) + 2 × (A + T)

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Tm = 81.5 + 16.6 × (log10 [Na+]) + 0.41 × (%G + C) – 675/n� 1. Codon Usage 2. Deoxyinosine

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1. Nearest neighbor analysis

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1. Beers Law

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133 #��� � � :"�J��F�

*'+,5-3: 4a�b U <) 5� ���! #&�</ � H��� L.

2M ���P� �� #6 2M �$2J4 �+3���

���* �+�* mM 1,000 mM = 1 molar (M) 1000mM/1M �21M/1000mM

�+�*'�8�* µM 1,000,000 µM = 1 M 1000µM /1mM �21mM/1000µM

• .�C� :500 �,1 .��<� �A��-0 2���2� �?E� �A��

�500 µM x 1 mM/1000 µM = 0.5 mM

• 04/0 ��2� �?E� �A��-0 2� �,1 �A��

�0.04 mM x 1000 µM/1 mM = 40 µM

$<:=� ���� ���W+ . c�.�� �5��" �d%e

��2� l��'� ��0H� ef5�-� - ���f�� ���f� �� DY 6� ���^�fE� �5 # ����� �)-�^�� �)�NE�P� ��4�� �t�(� �,(��� .#N,I 6� � ( ](� ��4��N(6 lf��'� :-� ](�f,1 6� [( 04 # ?E� ��2t�(�

;�a��� ���) - �02O ��0R ���^�E� ���� �0^,� X � [( X3 ��05 ���� [( e$H ��-�M " ?fE��" � ��b,� #5����� ��I- ?E�� X3 ��� #5 D�2E.

+(��6Y [( ��8�� �� ;�a� # �)F � 6� ���2B5 T2�fB4 #fI��� DY �f5 #NfE�P� #f5 c�f50�?R� j�� ;2 0) [( 0b� ���� ?b�a D��-Y ?E� #5 ��05 6�2� ���� ��4 +,f �- [( ��PCR

E�?. .��P� [( �.�C� D��,� #55 ��2� T(�2,��(0� �A��(MgCl2) T(��� D�0f #H�t� #5 6�2� - +, �- [( #5 �02�_ .��P� ](� 6� ��83200 +,f �- c�f�'� # ���S #5 ?E� �0�2M-0 2�

�(�� ?b�a �����100 6� �A�� -0 2�MgCl2 ���5 . ���f� #H�t� �(�5 �02�_ .��P� 6� ���$� #1 :-� -� #�LB� ](� X3 ��05 0�NE�,� #s��� �8,(� ���� ���:

� � �<�:

(Ci)(Vi) = (Cf)(Vf)� �� D�25 n�H .��0H #f2M-� �0f2�_ .��P� �� W0<� ?b�a # �, (Ci) Tf83 [f( �� \0ft

]2<� (Vi) �(�5 �(�� ?b�a �5 05�05 W0f<� ��f� z2R� (Cf) �(�f� Tf83 �� \0ft(Vf) +,f �-z2R� W0<� ���P� ���5 ��� .�05�5 #�LB� [( X3 � ]f(� 6� ���^�E� #fM��<� ���� �f(�5 ��f�5� � ��f4

��I����� k'� . ���� ��f�) #f ��� X/�3 D�,2�S� �(�5 ]2,&�4 �f���� #5�f� ��4�f3�- �f4 . #M��<� X3 6� XNR �(�5 ���/ ](� 02a ���3�- #5 #5�� ��4���� X(�N).

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134 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

]22<) 0(6 ���/ #5 ���� 04 .�C� ��05�� ���: Ci = 5 mM MgCl2�

Vi = unknown�

Cf = 100 µM MgCl2�

Vf = 200 µL� �3�- # �0 #I�) �(�5 ���� ](� ��Ci ��2� �3�- - �A��Cf ?E� �A��-0 2� .Ci X f� #f5

0(6 �� ���)��� X(�N) �A��-0 2� #5 �:

Ci = 5 mM MgCl2 × 1000 µM/1mM = 5000 µM MgCl2�

X3 X � ](� #5 #�LB� D�, � �� ���:

(5000 µM)(Vi) = (100 µM) (200 µL)� Vi = (100 µM)(200 µL)/5000 µM = 4 µL�

](�05�,54 6� 0�2M-0 2�MgCl2 5 ��2� +,f �- #f5 �(�5 �A��200 �f) ��f� #H�ft� �0f�2M-0 2� �(�� ?b�a100 �(Y ?E� #5 �A��-0 2�.

� 7� �<� :��LM i�� ���)� ���F41

[( .-� 02B� �� 022U) �5 #M��<� �f� f���) #�f��� � ?fb�a ��4�f3�- #f�4 #f ���fS #f5 ��f� X(�fN) ��f3�- #5 #M��<� o1 ?�E �� �f� ��f� #�f��� �(�f� ?fb�a �f3�- ��f,(��� #f ���f�

?E� #M��<� ?E�� ?�E �� . - ���f/ �� #f #fM��<� o1 ?�E �� ��� #���� ��4�3�- ����)W`3 - ��� ���E ����� ��I- 0B x0'� �� ) ���� �f��0O X(�fN) 0fb� ���f� ��f/� �3�- #5 � .

�,�B4 �A��<� 0(`��� �(��I +'5 �4�3�- X(�N) . XNR #�LB� #M��<� �� ���)��� #���� X � ](� #5 �:

5 mM MgCl2× 1000 µM/1 mM × X µL/200 µL = 100 µM MgCl2�

6�faY #f2M-� �0f2�_ ?fb�a �5 #M��<� # �2, #I�) �f� ��f� X(�fN) ��0f5 �f(�5 #f �� #f&�Y - �f� y0f� �E05 #M��<� ?E�� ?�E �� 0b� ���� �(�� ?b�a #5 �) ��� ��8�� �02�_ .��P� �f4� .

](� ?$2$3 ���� #M��<� 0f�2M-0 2� �f,1 �fE0�)X 0f�2M-0 2� (6� MgCl2 r,f� �f�2� �A�f� �f(�5 � #5 T83200 �(�f� ?fb�a #f5 �f) ��f� #H�t� 0�2M-0 2� MgCl2 �A�f�-0 2� �f/ 5 �fE0 . X(�fN)

1. Dimensional Analysis and Canceling Terms

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135 #��� � � :"�J��F�

��2� X(�fN) ;(0ft 6� ���^�E� �5 #M��<� �� �A��-0 2� #5 �A�� 1000 �A�f�-0 2�/1 �f�2� �A�f��� ���/ �02O .�3�- ����) �� W`3 #M��<� o1 ?�E �� �4 #5 �) ���� "�A��-0 2�" ,E05�.

• D��-Y ?E� #5X:

(5000 µM MgCl2)(X)/200 = 100 µM MgCl2�

X = (200)(100 µM MgCl2)/5000 µM MgCl2 = 20000/5000 = 4�

�5 .��P� ]���� ��05 ��NE�P� ](� #5 #I�)MgCl2 �(�5 �A��-0 2� �/4 .�f�P� 6� 0�2M-0 2� MgCl2 r,���2� #5 �A��200 ��� ��-�H� +, �- .��P� 0�2M-0 2�.

4���51 ��V-5�.

?E� �(�40�2�B�0)-��� 6� � ( ]2M� X2�E� ?fE� ��f� #f<M�Q� Xf�� ��fS #f5 #f . �fS �� #�fB � 6�0�fE� ]2M�f X2�E� T(��Y eE�) ]2M� X2�E� ��N�� #�� ) .�,�2E .�E�� �f� ��f� . DK

6�0�E� ]2M� X2�E�)ACHE( ���� .�fS 6�f5 ?f^I �4�/ # ?E� �O��5 DK . ](0f�) D��f,� #f5 ?b�a ?�BR ](� �� #<QR [( 02C ) ��b,� #5 ��(�2,� �(0� - 0�(�0� #,25 ��4553 �6�f5 ?f^I

�E�05 ���� ��I- -�O ���K �� # DK ](� 6��� ���. �� ��8�� D�2E�0�2) +, �- -� ��b,� ](� #5 �02O :W0<� 6� ��� 04 �(��H� 0(��$� #f5 �f(�5 �4

#M�M 6� �0E [( ��-�H� +, �- ��4���� . ��f(�2,� �f(0� ?b�a ���(�2,� D�2E�0�2) +(��6Y �� 6�5/0 �) �A��-0 2�10 ?fE� 02U�� �A��-0 2� . 6� 0f�(�0� ?fb�a ���f<5 +,f �- #f���8� ��05/0 �) �A��-0 2�2 �� 022U) �A��-0 2� �, .

W0<� 6� [( 04 T83 �(�5 ���5� �� +, �- �� 6�2� ���� ��4PCR ��f� #NE�P� .�� +,f �-�� 022U) ��(�2,� ?b�a - ?5�u 0�(�0� ?b�a ���(�2,� D�2E�0�2) �, . D�2fE�0�2) +,f �- �� ]2,&�4

�� 022U) 0�(�0� ?b�a - ?5�u ��(�2,��(0� ?b�a �0�(�0� �, . +f'5 #f5 ��NE�P� ](� ](�05�,5 ��f4 T2B$) ��8��� ��� .��/� c��'� #2) #5 6�2� ��NE�P� ��8�� 6� �<51 E��M��P� # ? #f�4 ��f�P�

02C ) ��N2 0)PCR )D�� #,25 ��05 0b� ���� ;2 0) - ��M� �I #5 ( ?fE� . c�f�'� ��f(��P� VifE �� �-�B� ��S #5 ��/�7 Jf(6�) +,f �- #M�M �f� ��f� . ]f(� 6� [f( 0f4 �� ?f^4 0(��f$� �#fM�M

���� D�� #,25 #5 6�2� # �N2 0) 6� �^��'�)0�(�0� �( ��(�2,� �(0� ( ��-�fH� �f� ��f� . �f,1 0f4 1. Master mix

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136 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

���^�E� ��/� c��'� #2) ��05 \Y�� ��� ?fE� 6�f2� +,f �- Tf83 ]���� #�� ?5�u ��b,� #5 � +, �- #5 ��� ��-�H� 02U�� ;2 0) 6� �0�� 0(��$� ���P� ��4 . ���M�DNA ](0f�Y D��,� #5

�� #H�t� #M�M 04 #5 ;2 0) ��0O . �(�5 +, �- 04 �(�� T8350 ���5 �A��-0 2�.

!"� 4F. $�B��C� : 4/��" $�B���; �. �+"��PCR

.��0H ��NE�P� ��8�� ��05(Ci)(Vi) = (Cf)(Vf) ?E� �2^�. Ci =#2M-� ?b�a Vi =#2M-� T83 Cf =�(�� ?b�a Vf =T83 �(�� "#2M-�" W0<� ?b�a - T83 #5 ��4 - �02�_"�(��" #5 ��05 0b� ���� ?b�a - T83 ��0O.

1- ��� � f��e!

• h� <� - -02� ��40�(�0� 6� ��� 04ACHE ?b�a50 ����� �A��-0 2� . #2) ��05 6� ���^�fE� �5 0�(�0� c��'�8 6� ���f 0f4 ?fb�a ��f40�(�0� 6� ���f 0f4 6� 0f�2M-0 2�

# 0�(�0� c��'� �� �40�(�0�16 �?E� ��$1 ���� T83 0�2M-0 2� Ci = 50 µM�

Cf = X� Vi = 8 µL (8 µL) (50 µM) = (X) (16 µL)�

Vf = 16 µL X = 25 µM�

0�(�0� c��'� �� �40�(�0� 6� ��� 04 ?b�a25 �A��-0 2� �� ���5. • +, �- #���8� �� ��4 ?b�a ���(�2,� �(0� D�2E�0�2)5/0 0f�(�0� 0f4 6� �A��-0 2�

�- [( �� +,50 ?E� 6�2� ���� �0�2M-0 2�. • ��$1 6� c��'� 0�(�0� �(�5 ���^�E� ��� �) ?b�a 0�(�0� �� 04 \�2)-0 2� #5 ](� ���$� ��E05

Ci = 25 µM� Cf = 0.5 µM�

Vi = X (25 µM)(X) = (0.5 µM)(50 µL) � Vf = 50 µL X = 1 µL�

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137 #��� � � :"�J��F�

+, �- [( ��50 ��0�2M-0 2�1 2M-0 2� ���^�fE� �f(�5 0f�(�0� c��'� 6� 0� ?fb�a �f) ��f� #5 �40�(�0� 6� [( 04 �(��5/0 �E05 �A��-0 2�.

�$��� �T�U1/0 '$ � 6 � � �e+� ?��

• ��� z2R� 0�(�0� c��'� 0�2M-0 2� �,1)20 ?b�a �5 ��� z2R� 05�0525/1 �A��-0 2� ( +, �- #550 +,f �- �� 0�(�0� 04 �(�� ?b�a �) ��� ��-�H� �0�2M-0 2�1/0 �A�f�-0 2�

���� Ci = 1.25 µM�

Cf = 0.1 µM�

Vi = X (1.25 µM)(X) = (0.1 µM)(50 µL)�

Vf = 50 µL X = 4 µL�

+, �- [( ��0550 ��0�2M-0 2�4 ��f� ���^�fE� �f(�5 ��� z2R� 0�(�0� c��'� 6� 0�2M-0 2�� ?b�a �) �(�1/0 �(Y ?E� #5 �A��-0 2�.

�$��� �T�U2/0 '$ � 6 � � �e+� ?��

• ��� z2R� 0�(�0� c��'� 0�2M-0 2� �,1)20 ?b�a �5 ��� z2R� 05�0525/1 �A��-0 2� ( +, �- #550 +,f �- �� 0�(�0� 04 �(�� ?b�a �) ��� ��-�H� �0�2M-0 2�2/0 �A�f�-0 2�

���� Ci = 1.25 µM�

Cf = 0.2 µM�

Vi = X (1.25 µM)(X) = (0.2 µM)(50 µL)�

Vf = 50 µL X = 8 µL� +, �- [( ��05 ](�05�,550 ��0�2M-0 2�8 ���^�fE� �f(�5 ��� z2R� 0�(�0� c��'� 6� 0�2M-0 2�

� ?b�a �) ��� �(�2/0 �(Y ?E� #5 �A��-0 2�. 0�(�0� ���b�a 0(�E ���� �� ;2)0) ]2�4 #5 -)5/0 �1 -2 �A�� -0 2� ( ��8�� ��NE�P� �f� ��f� ��0O #NE�P� �02�_ .��P� 6� 6�2� ���� T83 �).

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138 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

��b;: +, �- ��05 �0f2�_ 6� �f��5 6�f2� ���� 0�(�0� 6� �(A�5 ?b�a # �, �- 04 - ��<5 ��4 ���^�E� 0�(�0� ��/� �� ��� .X/�3 D�,2�S� #�2E- ](�5 �� ��� �f� ��-�fH� 0�(�0� T83 # #f5 �

# +, �- T83 6� +, �-50 0�25 ?E� 0�2M-0 2� ��� ��0O. 2- ����� DNA

+, �- #5 # ?E� ;2 0) ](0�Y A��<� ��M�PCR #H�t��� ��� . ?fb�a .�fC� ](� ��DNA ����K20 05 �0O-0 2���2� 0�2M �� ���5. • .�$��� ��05 �02�_ ](� 6� ��83 #1200 �0O����DNA +, �- #550 6�f2� �0f�2M-0 2�

�� # �2��5 #���� #I�) ����5200 ���� ?b�a #� - ?E� ���$� �0O����!

Ci = 20 µg/mL x mL/1000 µL x 1000 ng/µg = 20 ng/ µL� Cf = 200 ng/50 µL = 4 ng/µL�

Vi = X (20 ng/µL)(X) = (4 ng/µL)(50 µL)� Vf = 50 µL X = 10 µL �

+, �- ��05 ](�05�,550 ��0�2M-0 2�10 �02�_ 6� 0�2M-0 2�DNA ����K20 05 �0O-0 2���2� �) ��� ���^�E� �(�5 0�2M200 �0O����DNA ��0O +, �- ���-.

�:-: #H�ft� �-0O 04 eE�) ��� #���E ��/� c��'� #5 +, �- ���I� D��,� #5 ��<5 W0<� #E �� ���� .6 X(�B�#NE�P� +, �- [( ��05 0( �� ���� . c�f50� ��f� #f2) ��f/� c�f�'� Tf83 #58 �� +, �- ���5 . Tf83 XR��f3 6� 0f�� ?E� ] �� +, �- [( ��05 ��� #NE�P� ���$� #1 0O

�� ��NE�P� ](� ?(�� �� # �2��5 #���� 0S�� #5 ��� ���5 ��� 0�i�E eE�) ?���05 X5�R8 \0ft �� ��� ��� #H�t� ��/� c��'� #5 �). 3- .(%PCR

0H�f5 �02�_ .��P�PCR .��f<� �f�b�a10X ���� . +,f �- [f( ��50 ?fb�a �0f�2M-0 2� �0H�51X ?E� 6�2� ����. • +, �- [( ��0550 0H�5 ��$1 �0�2M-0 2�10X �(�f� ?b�a �) ��� ���^�E� �(�51X

����

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139 #��� � � :"�J��F�

Ci = 10X�

Cf = 1X� Vi = X (10X)(X) = (1X)(50 µL)�

Vf = 50 µL X = 5 µL� +, �- ��0550 ��0�2M-0 2�5 0H�f5 6� 0f�2M-0 2�PCR ?fb�a �f510X +,f �- c�f�'� #f5

��-�H� �� ��� �(�� ?b�a �)1X �(Y ?E� #5. 4- dNTP

�02�_ .��P�dNTP �� ?b�a10 ��2� ���� �A�� . �02�_ .��P� ](� ��4 q��dNTP ?fb�a �5 ���� ��I- 05�05 . 04 ](�05�,5dNTP )dATP, dCTP, dGTP -dTTP ( .��<� ��b�a5/2 ��2� �A��

���� . +, �- 04 ��50 6� q�� 04 ?E� 6�2� �0�2M-0 2�dNTP ?fb�a �5 �4200 ��fI- �A�f�-0 2� ���5 #����.

• �02�_ .��P� 6� ��$1dNTP ?b�a �510 ��2� �f) ��f� ��-�H� �(�5 +, �- 04 #5 �A�� q�� 04 ?b�adNTP .�<�200 ���� �A��-0 2�

Ci = 2.5 mM x 1000 µM/mM = 2500 µM� Cf = 200 µM�

Vi = X (2500 µM)(X) = (200 µM)(50 µL)�

Vf = 50 µL X = 4 µL�

+, �- [( ��0550 ��0�2M-0 2�4 �02�_ .��P� 6� 0�2M-0 2�dNTP ?fb�a �f510 �A�f�-0 2� 04 �(�� ?b�a �) ��� ���^�E� �(�5dNTP .��<�200 ��� �A��-0 2�.

5- Taq ��� �� !

T(��Y �02�_ .��P�Taq ��� 6�0� .��<� ��b�a5 0�2M-0 2� 05 �3�-)Units/ µL (���� . ��0f5 +, �- 0450 6� ���^�E� #5 6�2� �0�2M-0 2�25/1 �� T(��Y �3�- ���5.

• 0 `) ���� #H�t� #M�M 04 #5 �(�5 ��(��Y �02�_ .��P� 6� ��$1 :25/1 ���$� [( �3�-?b�a #� ?E�.

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140 ����� ��� �� ���� ���� � ���� � ��� � �(PCR)

Ci = 5 Units/µL�

Cf = 1.25 Units/50 µL = 0.025 Units/µL� Vi = X (5 Units/µL)(X) = (0.025 Units/µL)(50 µL)�

Vf = 50 µL X = 0.25 µL� +, �- [( ��0550 �(�5 �0�2M-0 2�25/0 6� 0�2M-0 2�Taq ��� 6�0� ?fb�a �f55 0f5 �f3�-

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Page 177: م د - mums.ac.ir...Amplification Refractory Mutations System-PCR 0ff2C TˇffB2E 6 0ff ff ˛ 0 ff28 6 +,ff -+ I ,, V

Mashhad University of Medical Sciences

Vice Chancellor for Research

Publication No. 406

Applied Optimization of

Polymerase Chain Reaction (PCR)

By:

Dr. Khadijeh Jamialahmadi, Ph.D.

School of Medicinc and Biotechnology Research Center

Mashhad University of Medical Sciences

Fahimeh Afzaljavan, M.Sc.

Ph.D. Student of Molecular Medicine

School of Medicine, Mashhad University of Medical Sciences