14 Polymerase Chain Reaction
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• Labeled primers can be employed for many purposes. Biotinylated primers can be used for purification of PCR products using streptavidin
coated magnetic beads. Fluorescence-labeled primers can be used for
automated detection of PCR products.
• Multiplex PCR is the coamplification of several PCR products using several primer pairs in the same reaction mixture.
Specific cycling protocols
• Asymmetrie PCR describes one approach to overcome the problern of
shadow bands resulting from the opposite PCR primer in direct sequencing of a PCR product (41).
• "Hot PCR" is used to incorporate a radioactive label during PCR (e.g. a[32P]-dCTP) during PCR).
Allele detection and sequencing of PCR products
• Allele-specific oligonucleotides (ASOs) describes the use of labeled oligonucleotides for differential dot blot hybridization to PCR products
that exhibit point mutations (42, 43, 44, 45).
• Reverse dot blot method is the reverse of the ASO method: the labeled
PCR product is hybridized to several dot blotted ASOs (46).
• Denaturing gradient gel electrophoresis (DGGE) (47, 48) and temperature gradient gel electrophoresis (TGGE) are used to discriminate heteroduplexes and homoduplexes of PCR products from templates containing point mutations (see Chapter 6).
• Single-strand conformational polymorphism (SSCP) allows for discrimination of point mutations by differential migration of single strands in
electrophoresis (49) (see Chapter 6).
• Oligomer ligation assay ( OLA) is a technique for allelic discrimination by
use of a heat-stable ligase (50).
• Restrietion enzyme digestion of PCR products can be used for allele discrimination of loss or addition of a restrietion enzyme recognition site.
• Direct sequencing ofPCR products has been described in many protocols
(51) all aimed at avoiding "shadow sequence" derived from the second PCR
primer.
221
• Labeled primers can be employed for many purposes. Biotinylated primers can be used for purification of PCR products using streptavidin
coated magnetic beads. Fluorescence-labeled primers can be used for
automated detection of PCR products.
• Multiplex PCR is the coamplification of several PCR products using several primer pairs in the same reaction mixture.
Specific cycling protocols
• Asymmetrie PCR describes one approach to overcome the problern of
shadow bands resulting from the opposite PCR primer in direct sequencing of a PCR product (41).
• "Hot PCR" is used to incorporate a radioactive label during PCR (e.g. a[32P]-dCTP) during PCR).
Allele detection and sequencing of PCR products
• Allele-specific oligonucleotides (ASOs) describes the use of labeled oligonucleotides for differential dot blot hybridization to PCR products
that exhibit point mutations (42, 43, 44, 45).
• Reverse dot blot method is the reverse of the ASO method: the labeled
PCR product is hybridized to several dot blotted ASOs (46).
• Denaturing gradient gel electrophoresis (DGGE) (47, 48) and temperature gradient gel electrophoresis (TGGE) are used to discriminate heteroduplexes and homoduplexes of PCR products from templates containing point mutations (see Chapter 6).
• Single-strand conformational polymorphism (SSCP) allows for discrimination of point mutations by differential migration of single strands in
electrophoresis (49) (see Chapter 6).
• Oligomer ligation assay ( OLA) is a technique for allelic discrimination by
use of a heat-stable ligase (50).
• Restrietion enzyme digestion of PCR products can be used for allele discrimination of loss or addition of a restrietion enzyme recognition site.
• Direct sequencing ofPCR products has been described in many protocols
(51) all aimed at avoiding "shadow sequence" derived from the second PCR
primer.