PCR Machine

Tools in Genetic
Engineering
Objectives:
At the end of the session the students should be able
to:
• Name the different tools in genetic engineering,
• Describe the function of each tool used in genetic
engineering; and
• Identify the different restriction enzymes used in
genetic engineering.
• Do you have any idea about PCR
machine?
• Have you seen it in picture or have
you seen it in foreground?
• Have you ever used a PCR machine?
 What is a PCR Machine?
• The thermal cycler (also known as
thermocycler, PCR machine or DNA
amplifier) is a laboratory apparatus most
commonly used to amplify segments of
DNA via the Polymerase Chain Reaction
(PCR).
 What is a PCR Machine?
• Thermal cyclers may also be used in
laboratories to facilitate other
temperature-sensitive reactions,
including restriction enzyme digestion or
rapid diagnostics.
 Polymerase Chain Reaction (PCR)

A technique used to “amplify” small segments

of DNA using a PCR machine.
 The purposes of a PCR machine:

•  Gene cloning, sequencing of complex

genomes, DNA fingerprinting and DNA-
based diagnostics are just some of the
techniques that PCR machines can do. 
The purposes of a PCR machine:
 Kary Mullis
• Kary Mullis is generally credited
with inventing PCR in 1983 while
working for Cetus Corporation in
Emeryville, California.
• Mullis received the Nobel Prize for
his ground-breaking invention in
1993.
PARTS OF A PCR MACHINE
 How does a PCR work?
Step 1. Denaturation
Step 2. Annealing
Step 3. Extension / Elongation
 Denaturation (94-95°C)

• Denaturation is the alteration of a protein

shape through some form of external stress
(for example, by applying heat, acid or alkali),
in such a way that it will no longer be able to
carry out its cellular function.
 Annealing (54-55°C)
• Annealing is a heat treatment that alters the
physical and sometimes chemical properties
of a material to increase its ductility and
reduce its hardness, making it more
workable.
 Annealing (55°C)
• It involves heating a material above its 
recrystallization temperature,
maintaining a suitable temperature for a
suitable amount of time, and then
cooling.
 Synthesizing/ Extension (72°C)

• DNA synthesis is the process whereby

deoxynucleic acids (adenine, thymine,
cytosine, and guanine) are linked
together to form DNA.
 Variants of PCR
• Real-time PCR
• Competitive PCR
• PCR insitu
• RT-PCR( Reverse Transcriptase-PCR)
TOOLS USED IN
PCR
 Thermal Cycler
• Is also known as the PCR machine , a laboratory apparatus most
commonly used “to amplify” – meaning to increase segments of DNA
by the polymerase chain reaction. The apparatus can also facilitate
sensitive reactions that required a high and low temperature such as
restriction enzyme digestion and rapid results diagnosis.
 Centrifuge
Is an instrument that can separate component parts a liquid or
fluid by using centrifugal force that will help in spinning the
fluid at high speed within a container. The liquids separated
from the solid or can be separation of fluids of different
densities.
 Transilluminator
A highly delicate piece of equipment which works
through the emission of high levels of ultraviolet
radiations. It is used in life laboratories for the
process of interpreting in visual terms the target
DNA and proteins through the viewing surface.
 LESSON 3

Restriction Enzymes as a Tool

( Concepts and Process)
 Bacterias present in PCR
process:
• Taq polymerase - enzyme originally isolated
from the thermophilicbacterium 
Thermus aquatic.
• E coli – a bacteria usuallly identified by a PCR
machine.
 What are enzymes?
Enzymes are biological catalysts-found in all cells.
They are molecules ( usually proteins) that speed
up chemical reactions. They work critically for a
range of cellular processes including digestions,
DNA replication, and protein synthesis.
How about the so called restriction enzymes?

- Restriction endonucleases
- Restriction enzymes are termed as “
molecular scissors”
Why molecular scissors?
The restriction enzymes have the
ability to recognize specific base-pair
sequences of DNA, and then cut the
double- stranded DNA at those sites.
Why researchers rely on restriction enzymes?

1. Making recombinant DNA and appraising

success.( research, Medicine & agriculture)
2. DNA analysis.( relationship testing & forensics)
3. Used in gene cloning.
4. Protein expression experiments.
 Some terms associated with
restriction enzymes
Recognition site -  or restriction
recognition sites, are located on a DNA
molecule containing specific sequences of
nucleotides, which are recognized by
restriction enzymes.
A restriction site is a sequence of approximately 6–8 base
pairs of DNA that binds to a given restriction enzyme.
 Sticky ends
• DNA ends refer to the properties of the end of DNA molecules,
which may be sticky or blunt based on the enzyme which cuts the
DNA.
•  the ends left have one strand overhanging the other to form a
short (typically 4 nt) single-stranded segment.
• This overhang will easily re-attach to other ends like it, and are
thus known as "Sticky ends".
Blunt ends - no overhang
What I have learned?
What is PCR stand for?
Ans. Polymerase Chain
Reaction
 2. What are the steps involved in PCR?

Answer.
Step 1: Denaturing
Step 2: Annealing
Step 3: Elongation/Extension/Synthesizing
 3. It refers to a molecular scissors and
has the ability to recognize the specific
base-pair sequence in DNA.

Answer: Restriction enzymes / Restriction

endonucleases
What are the other term associated with
restriction enzymes?
a. Recognition site
b. Sticky ends
c. Blunt ends
 WHAT’S YOUR TASK?
For Quarter 3 Module 1 answer only the following parts/pages
- What I know: pages 1-3
- Independent assessment 1: pages 12-13
- What I can do / Performance task: page 16
- Assessment: pages 16-17
 END OF
PRESENTATION