Chapter 8.

0
Recombinant DNA Technology
8.2 Method in gene cloning

Recombinant DNA technology

Definition: Advantages:

Technique

Gene Cloning PCR DNA Fingerprinting

- DNA profiling
1. Host cell - DNA sequencing
2. Vector
3. Gene of interest Definition Uses of DNA fingerprinting
Tools involve 4. Restriction Enzyme
5. Modifying Enzyme

1. Isolation
2. Cleft
Mechanism 3. Insert
4. Transformation & Amplification Mechanism
5. Screening

Example in Agriculture, Production of Insulin cDNA; Transgenic animal

Example in Medical, Production of transgenic plant Ti plasmic; transgenic animal; GMO

Example in Environment, Role of transgenic bacteria; transgenic plant

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 Chapter 8.0
Recombinant DNA Technology
8.2 Method in gene cloning

Others technique in recombinant DNA technology

Polymerase
Chain
Reaction

A technique by which any piece of DNA can be quickly

amplified without using cells, through a series of
enzymatic reaction in test tube.
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 Chapter 8.0
Recombinant DNA Technology
8.2 Method in gene cloning

WHY need to carry out PCR

Produce a large amount of

identical copies of DNA
fragment in short time

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 Chapter 8.0
Recombinant DNA Technology
8.2 Method in gene cloning

COMPONEN IN PCR

1. DNA containing the

nucleotide sequence
2. Short sequences of DNA
called primers
Signal to polymerase to initiate synthesis of
DNA strand

3. Taq polymerase
4. Free DNA nucleotides

Recall: What is Taq polymerase? Why it is better than DNA polymerase?

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 Chapter 8.0
8.2 Method in gene cloning

Polymerase Chain Reaction: STEP

Recombinant DNA Technology

(1) Denaturing
(2) Annealing
(3) Extension

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 Chapter 8.0
Recombinant DNA Technology
8.2 Method in gene cloning

Step 1: Denaturing

DNA is separated into single

strands by heating to about 90oC-
96oC.

Force the break of hydrogen bond

and separation of DNA strand by
heat. [alternative method, no
enzyme use]

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 Chapter 8.0
Recombinant DNA Technology
8.2 Method in gene cloning

Step 2 : Annealing

1. Temperature is lowered to
50oC 55oC
2. to allow primers to form
hydrogen bond with
complementary bases at
opposite ends of target
sequence

Primer Role:
Signal to polymerase to initiate synthesis
of DNA strand

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Recombinant DNA Technology
8.2 Method in gene cloning

Step 3 : Extension
1. Temp. raise to 72C
2. Taq polymerase joins up the
nucleotides, beginning at the ends
of primers
3. synthesizing new DNA strands
that are complementary to those
original DNA

No DNA polymerase because

Taq-polymerase is resistance to
heat. Normal DNA polymerase
will denature at 72C
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 Chapter 8.0
8.2 Method in gene cloning

Polymerase Chain Reaction

Recombinant DNA Technology

Number of DNA produced =2

n
# After each cycle, the amount of
the target DNA doubles.

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Recombinant DNA Technology
DNA fingerprinting

DNA FINGERPRINTING

1. Study the chemical structure of DNA

2. Important in DNA profiling and gene
mapping

APPLICATION OF DNA FINGERPRINTING

1. Parent-child VNTR pattern analysis
2. Crime scene investigation suspect identification
3. Forensics identification
4. Personal identification diagnose inherited disorder

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