PCR APPLICATION Flashcards
WHAT IS PCR?
PCR IS AN IN VITRO METHOD USED FOR RAPID REPRODUCTION OF VERY LARGE AMOUNTS OF SPECIFIC DNA SEGMENTS
STATE THE PCR PRINCIPLE.
- IT IS AN IN VITRO REPLICATION OF SPECIFIC DNA SEQUENCES USING DNA POLYMERASE
- CAN GENERATE DNA FRAGMENTS FROM DNA EXTRACT
- IF THE DNA SEQUENCE OF INTEREST IS PRESENT IN THE DNA EXTRACT, IT IS POSSIBLE TO SELECTIVELY REPLICATE IT IN VERY LARGE NUMBERS
STATE THE 5 COMPONENTS OF PCR COMPONENTS.
- DNA TEMPLATE
- PRIMERS
- DNA POLYMERASE
- DNTPS
- BUFFER SOLUTION
BRIEFLY DESCRIBE THESE 2 PCR COMPONENTS:
1. DNA TEMPLATE
2. PRIMERS
- DNA TEMPLATE
- DNA TEMPLATE HAVE THE REGION OF THE DNA SEQUENCE OR TARGET SEQUENCE THAT WANTED TO BE AMPLIFIED.
- IT IS THE PRODUCT OF THE DNA EXTRACTION
- EXAMPLE: HAIR ROOTS, NAILS - PRIMERS
- PRIMERS IS THE OLIGONUCLEOTIDES THAT DEFINE THE SEQEUNCE TO BE AMPLIFIED.
- THE FUNCTION OF THIS PRIMERS IS TO ANNEAL THE SINGLE STRAND TEMPLATE.
- THERE IS TWO TYPES OF PRIMERS WHICH ARE FORWARD PRIMERS AND REVERSE PRIMERS.
- FORWARD PRIMERS IS USED TO ANNEAL THE ANTISENSE STRAND
- AS FOR THE REVERSE PRIMERS, IT IS TO ANNEAL THE SENSE STRANDS. IT WILL PROVIDE THE INITIATION SITE FOR EXTENSION OF NEW DNA.
BRIEFLY DESC THESE PCR COMPONENTS:
1. DNA POLYMERASE
2. BUFFER SOLUTION
1.DNA POLYMERASE
- DNA POLYMERASE SUCH AS THE TAQ POLYMERASE IS USED TO CATALYSED THE PCR REACTION.
- IT WILL ENTEND THE NEW DNA STRAND COMPLEMENT TO THE DNA TEMPLATE.
- BUFFER SOLUTION
- BUFFER SOLUTION CONTAIN THE ION AND SALT SUCH AS MAGNESIUM ION AND MANGANESE ION.
- THIS BUFFER IS USED TO MAINTAIN THE PH AND THE IONIC STRENGTH OF THE REACTION SOLUTION SO THAT THE DNA POLYMERASE CAN FUNCTION.
WHAT IS DNTPS?
DNTPS IS THE BULDING BLOCK FOR THE NEW STRAND OF DNA.
IT CONTAINS DATP, DTTP, DCTP, DGTP.
HOW TO AVOID CONTAMINATION DURING PCR CYCLE.
- SEPERATE THE AREAS FOR PCR AND DNA EXTRACTION
- USE GLOVE AND STERILE TIPS
- USE CLEAN PIPETTER
- ADDING CONTROL REACTION
- USE APPROPRIATE PCR CYCLING CONDITIONS
WHAT IS MEANT BY POSITIVE CONTROL?
POSITIVE CONTROL IS WHERE THE PCR TEMPLATE IS THE NUCLEIC ACID OF THE SAMPLE. THE PRECENCE OF THIS PCR BAND WILL VERIFY THE PRESENCE OF THE SAMPLE OR DIAGNOSIS.
WHAT IS MEANT BY NEGATIVE CONTROL?
NEGATIVE CONTROL IS A CONTROL WHERE THERE IS NO DNA TEMPLATE. THE RESULT SHOULD NOT HAVE ANY PCR BAND. THE PRESENCE OF PCR BAND INDICATE THAT THERE ARE CROSS CONTAMINATION TO THE REACTION MIXTURE.
THE CHOICE OF THE DURATION, THE ANNEALING TEMPERATURE AND THE NUMBER OF CYLCES ARE DEPENDS ON ___ AND ___
THE SIZE OF THE SEQUENCE OF INTEREST AS WELL AS THE SIZE AND THE COMPLEMENTARITY OF THE PRIMERS
WHY THE DURATION OF AMPLIFICATION NEED TO BE REDUCED TO THE MINIMUM?
TO SAVE TIME AND PREVENT RISK OF NON- SPECIFIC AMPLIFICATION.
WHAT IS THE BEST TEMPERATURE FOR DENATURATION?
94 - 96 DEGREE
WHAT IS THE BEST TEMPERATURE FOR ANNEALING?
60 DEGREE
WHAT IS THE BEST TEMPERATURE FOR EXTENSION?
72 DEGREE
STATE THE STEPS TO PREPARE THE AGAROSE GEL.
- PREPARE THE BUFFER SOLUTION
- ADDING THE AGAROSE GEL INTO THE BUFFER SOLUTION
- HEAT THE AGAROSE MIXTURE
- SWIRL THE AGAROSE MIXTURE SO IT IS PROPERLY DISSOLVED
- PREPARE THE GEL MOULD ASSEMBLY TOGETHER WITH THE COMB TO FORM THE WELLS.
- POUR THE DISSOLVED AGAROSE INTO THE GEL MOULD ASSEMBLY.
- REMOVE THE GEL MOULD ASSEMBLY ONCED THE GEL IS SOLIDIFIED.
