PCR APPLICATION

Question 1

WHAT IS PCR?

Answer 1

PCR IS AN IN VITRO METHOD USED FOR RAPID REPRODUCTION OF VERY LARGE AMOUNTS OF SPECIFIC DNA SEGMENTS

Question 2

STATE THE PCR PRINCIPLE.

Answer 2

1. IT IS AN IN VITRO REPLICATION OF SPECIFIC DNA SEQUENCES USING DNA POLYMERASE
2. CAN GENERATE DNA FRAGMENTS FROM DNA EXTRACT
3. IF THE DNA SEQUENCE OF INTEREST IS PRESENT IN THE DNA EXTRACT, IT IS POSSIBLE TO SELECTIVELY REPLICATE IT IN VERY LARGE NUMBERS

Question 3

STATE THE 5 COMPONENTS OF PCR COMPONENTS.

Answer 3

1. DNA TEMPLATE
2. PRIMERS
3. DNA POLYMERASE
4. DNTPS
5. BUFFER SOLUTION

Question 4

BRIEFLY DESCRIBE THESE 2 PCR COMPONENTS:
1. DNA TEMPLATE
2. PRIMERS

Answer 4

1. 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
2. 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.

Question 5

BRIEFLY DESC THESE PCR COMPONENTS:
1. DNA POLYMERASE
2. BUFFER SOLUTION

Answer 5

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.

2. 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.

Question 6

WHAT IS DNTPS?

Answer 6

DNTPS IS THE BULDING BLOCK FOR THE NEW STRAND OF DNA.
IT CONTAINS DATP, DTTP, DCTP, DGTP.

Question 7

HOW TO AVOID CONTAMINATION DURING PCR CYCLE.

Answer 7

• SEPERATE THE AREAS FOR PCR AND DNA EXTRACTION
• USE GLOVE AND STERILE TIPS
• USE CLEAN PIPETTER
• ADDING CONTROL REACTION
• USE APPROPRIATE PCR CYCLING CONDITIONS

Question 8

WHAT IS MEANT BY POSITIVE CONTROL?

Answer 8

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.

Question 9

WHAT IS MEANT BY NEGATIVE CONTROL?

Answer 9

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.

Question 10

THE CHOICE OF THE DURATION, THE ANNEALING TEMPERATURE AND THE NUMBER OF CYLCES ARE DEPENDS ON ___ AND ___

Answer 10

THE SIZE OF THE SEQUENCE OF INTEREST AS WELL AS THE SIZE AND THE COMPLEMENTARITY OF THE PRIMERS

Question 11

WHY THE DURATION OF AMPLIFICATION NEED TO BE REDUCED TO THE MINIMUM?

Answer 11

TO SAVE TIME AND PREVENT RISK OF NON- SPECIFIC AMPLIFICATION.

Question 12

WHAT IS THE BEST TEMPERATURE FOR DENATURATION?

Answer 12

94 - 96 DEGREE

Question 13

WHAT IS THE BEST TEMPERATURE FOR ANNEALING?

Answer 13

60 DEGREE

Question 14

WHAT IS THE BEST TEMPERATURE FOR EXTENSION?

Answer 14

72 DEGREE

Question 15

STATE THE STEPS TO PREPARE THE AGAROSE GEL.

Answer 15

1. PREPARE THE BUFFER SOLUTION
2. ADDING THE AGAROSE GEL INTO THE BUFFER SOLUTION
3. HEAT THE AGAROSE MIXTURE
4. SWIRL THE AGAROSE MIXTURE SO IT IS PROPERLY DISSOLVED
5. 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.

Question 16

STATE THE TYPES OF GEL THAT IS COMMON TO USE AND BRIEFLY DESCRIBE.

Answer 16

1. AGAROSE GEL
   - TO SEPERATE LARGE FRAGMENTS IN A LOW RESOLVING POWER.
   - IT CAN SEPERATE FRAGMENTS OF DNA FROM ABOUT 200 BP TO 50K BP
2. POLYACRYLAMIDE GEL
   - TO SEPERATE FRAGMENTS THAT IS LESS THAN 500 BP.

Question 17

WHAT IS THE EXAMPLE OF BUFFER SOLUTION?

Answer 17

TBE AND EDTA

Question 18

STATE THE STEPS OF GEL ELECTROPHORESIS.

Answer 18

1. MOUNT THE AGAROSE GEL IN THE ELECTROPHORESIS TANK
2. MIX THE PCR LADDER AND PRODUCTS WITH THE LOADING DYE
3. PIPETTE THE PCR LADDER AND PRODUCTS INTO THE WELLS
4. APPLY THE ELECTRIC CURRENT
5. SMALL SIZE PRODUCT WILL MIGRATE TO THE ANODE TERMINAL MUCH FASTER.
6. STOP THE ELECTRIC CURRENT WHEN THE LADDER/ PRODUCT WELL SEPERATED.

Question 19

STATE THE DNA DYE THAT CAN BE USED TO VISUALISED THE PCR PRODUCTS.

Answer 19

ETHIDIUM BROMIDE AND SYBR GREEN

Question 20

STATE THE COMMON TYPES OF PCR.

Answer 20

1. ALLELE SPECIFIC PCR
2. REVERSE TRANSCRIPTASE PCR
3. MULTIPLEX PCR
4. NESTED PCR
5. REAL TIME PCR
6. SINGLE CELL PCR

Question 21

STATE THE MEDICAL APPLICATION OF PCR.

Answer 21

1. TO DETECT THE HERIDETARY DISEASE
2. FOR PATERNAL TESTING
3. DNA PROFILLING
4. DIAGNOSIS OF INFECTIOUS DISEASE
5. CLONING OF GENES