Contents

• Important Note
• Copyright
• Introduction
• Installation Instructions
• System Requirements
• Input and Output Conventions
• ``Sequence'' Input Tags
  • PRIMER_SEQUENCE_ID
  • SEQUENCE
  • INCLUDED_REGION
  • TARGET
  • EXCLUDED_REGION
  • PRIMER_COMMENT
  • COMMENT
  • PRIMER_SEQUENCE_QUALITY
  • PRIMER_LEFT_INPUT
  • PRIMER_RIGHT_INPUT
  • PRIMER_START_CODON_POSITION
  
  
• ``Global'' Input Tags
  • PRIMER_PICK_ANYWAY
  • PRIMER_MISPRIMING_LIBRARY
  • PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS
  • PRIMER_MAX_MISPRIMING
  • PRIMER_MAX_TEMPLATE_MISPRIMING
  • PRIMER_PAIR_MAX_MISPRIMING
  • PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING
  • PRIMER_PRODUCT_MAX_TM
  • PRIMER_PRODUCT_MIN_TM
  • PRIMER_EXPLAIN_FLAG
  • PRIMER_PRODUCT_SIZE_RANGE
  • PRIMER_PICK_INTERNAL_OLIGO
  • PRIMER_GC_CLAMP
  • PRIMER_OPT_SIZE
  • PRIMER_DEFAULT_SIZE
  • PRIMER_MIN_SIZE
  • PRIMER_MAX_SIZE
  • PRIMER_OPT_TM
  • PRIMER_MIN_TM
  • PRIMER_MAX_TM
  • PRIMER_MAX_DIFF_TM
  • PRIMER_MIN_GC
  • PRIMER_OPT_GC_PERCENT
  • PRIMER_MAX_GC
  • PRIMER_SALT_CONC
  • PRIMER_DNA_CONC
  • PRIMER_NUM_NS_ACCEPTED
  • PRIMER_SELF_ANY
  • PRIMER_SELF_END
  • PRIMER_DEFAULT_PRODUCT
  • PRIMER_FILE_FLAG
  • PRIMER_MAX_POLY_X
  • PRIMER_LIBERAL_BASE
  • PRIMER_NUM_RETURN
  • PRIMER_FIRST_BASE_INDEX
  • PRIMER_MIN_QUALITY
  • PRIMER_MIN_END_QUALITY
  • PRIMER_QUALITY_RANGE_MIN
  • PRIMER_QUALITY_RANGE_MAX
  • PRIMER_INSIDE_PENALTY
  • PRIMER_OUTSIDE_PENALTY
  • PRIMER_MAX_END_STABILITY
  • PRIMER_PRODUCT_OPT_TM
  • PRIMER_PRODUCT_OPT_SIZE
  • PRIMER_TASK
  • PRIMER PENALTY WEIGHTS AND PRIMER PAIR WEIGHTS
  
  
• Internal Oligo ``Sequence'' Input Tags
  • PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION
  • PRIMER_INTERNAL_OLIGO_INPUT
  
  
• Internal Oligo ``Global'' Input Tags
  • PRIMER_INTERNAL_OLIGO_OPT_SIZE
  • PRIMER_INTERNAL_OLIGO_MIN_SIZE
  • PRIMER_INTERNAL_OLIGO_MAX_SIZE
  • PRIMER_INTERNAL_OLIGO_OPT_TM
  • PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT
  • PRIMER_INTERNAL_OLIGO_MIN_TM
  • PRIMER_INTERNAL_OLIGO_MAX_TM
  • PRIMER_INTERNAL_OLIGO_MIN_GC
  • PRIMER_INTERNAL_OLIGO_MAX_GC
  • PRIMER_INTERNAL_OLIGO_SALT_CONC
  • PRIMER_INTERNAL_OLIGO_DNA_CONC
  • PRIMER_INTERNAL_OLIGO_SELF_ANY
  • PRIMER_INTERNAL_OLIGO_MAX_POLY_X
  • PRIMER_INTERNAL_OLIGO_SELF_END
  • PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY
  • PRIMER_INTERNAL_OLIGO_MAX_MISHYB
  • PRIMER_INTERNAL_OLIGO_MAX_TEMPLATE_MISHYB
  • PRIMER_INTERNAL_OLIGO_MIN_QUALITY
  • INTERNAL PRIMER PENALTY WEIGHTS
  
  
• Example
• Output Tags
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO | PAIR}_<tag_name>.
  • PRIMER_ERROR = <string>(*)
  • PRIMER_LEFT = <start>,<length>
  • PRIMER_RIGHT = <start>,<length>
  • PRIMER_INTERNAL_OLIGO = <start>,<length>
  • PRIMER_PRODUCT_SIZE = <int>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_EXPLAIN = <string>(*)
  • PRIMER_PAIR_EXPLAIN = <string>(*)
  • PRIMER_PAIR_PENALTY = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_PENALTY = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_SEQUENCE = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_TM = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_GC_PERCENT = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_SELF_ANY = <float>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_SELF_END = <float>
  • PRIMER_PAIR_COMPL_ANY = <float>
  • PRIMER_PAIR_COMPL_END = <float>
  • PRIMER_WARNING = <string>(*)
  • PRIMER_{LEFT | RIGHT | PAIR}_MISPRIMING_SCORE = <float>, <string>
  • PRIMER_{LEFT | RIGHT | PAIR}_TEMPLATE_MISPRIMING = <float>
  • PRIMER_PRODUCT_TM = <float>
  • PRIMER_PRODUCT_TM_OLIGO_TM_DIFF = <float>
  • PRIMER_PAIR_T_OPT_A = <float>
  • PRIMER_INTERNAL_OLIGO_MISHYB_SCORE = <float>, <string>
  • PRIMER_{LEFT | RIGHT | INTERNAL_OLIGO}_MIN_SEQ_QUALITY = <int>
  • PRIMER_LEFT,RIGHT_END_STABILITY = <float>
  • PRIMER_STOP_CODON_POSITION= <int>
  
  
• Example Output
• Advice for Picking Primers
• Cautions
• What to do if Primer3 Cannot Find Primers?
• Differences from Earlier Versions
• Exit Status Codes
• The New Primer3 WWW Interface
• Acknowledgments