Recombinant DNA Technology

Objectives:
 Compare classical breeding with modern

genetic engineering techniques
 Outline the steps involved in genetic

engineering
SHS General Biology 2 All Rights Reserved

© Oxford Publishing (Malaysia) Sdn. Bhd. (1175094-D), 2018 1– 1
6
MODERN
GENETICS
Recombinant DNA technology
 Mendelian genetics and modern genetics are really parts of the

same thing. Gregor Mendel formed the basis of modern genetics.
Later scientists built on his ideas and laws, elaborating on them.
Nothing in modern genetics disagrees with Mendel's interpretation
of genetics, but it has found cases where genetics is more
complicated than the version he uncovered.
 Genetic engineering (or genetic modification) is the process of
altering the DNA in an organism’s genome and hence alter the
organism’s characteristics (phenotype) in a particular way.
 This may involve: changing one base pair (A-T or C-G), deleting a
whole region of DNA, or introducing an additional copy of a gene. It
may also mean extracting DNA from another organism’s genome
and combining it with the DNA of that individual.

Uses of genetic engineering
 Can be used to produce plants that have a

higher nutritional value or is able to resist
parasitic attacks or can tolerate exposure to
herbicides, etc.
 Has been applied to improve the resilience,
nutritional value and growth rate of crops, e.g.
rice, tomatoes, potatoes.

Classical breeding
 Classical breeding or selective breeding is a form of genetic

modification that does not involve the addition of any foreign DNA
into the organism. Instead, it is the planned selection of desirable
traits.
 Breeders select 2 parents that have the desired phenotypic traits to
reproduce, yielding offspring with those desired traits. Z
 Without selective breeding, many plants and animals today would
not exist.
 Inbreeding is one of the disadvantages of classical breeding.
Through inbreeding, 2 closely related organisms reproduce to yield
a purebred with desired traits. But these organisms may also have
undesirable traits due to recessive genes found in both parents.

Recombinant DNA (rDNA)
Technology
 Technique used in genetic engineering that

involves the identification, isolation and
insertion of “desired” gene (target DNA) into a
vector such as a plasmid or bacteriophage to
form a recombinant DNA molecule and
production of large quantities of that gene
fragment or product encoded by that gene.
Example: humulin, is insulin developed using
rDNA technology that is used to treat diabetes.
Tools Used in Recombinant
DNA Technology
 target DNA
 restriction enzymes
 DNA cloning vectors
 host cell
 modifying enzymes

Where do we get this gene of
interest?
 Genomic library
 cDNA library
 Chemical synthesis of gene if we know the
sequence
 if the number of copies of the desired gene is
not enough for gene cloning we can opt for
gene amplification techniques like PCR

Steps in rDNA technology
 Step 1: Select and isolate of target DNA (aka “gene of interest”)

Selection of DNA segment to be cloned. This segment is then isolated
enzymatically. This segment is termed as target DNA.
 Step 2: Select a suitable cloning vector
A cloning vector is a self-replicating DNA molecule, into which the target
DNA is to be integrated. A suitable cloning vector (e.g. plasmids or
bacteriophages) needs to be selected.
 Step 3: Introduce target DNA into vector to form rDNA molecule
The target DNA which has been extracted and cleaved enzymatically by
the selective restriction endonuclease enzymes (in Step 1) are then
ligated (joined) by the enzyme ligase to vector DNA to form a rDNA
molecule.

Steps in rDNA technology
(cont’d)
 Step 4: rDNA molecule is introduced to a suitable host
Suitable host cells selected and the rDNA molecule formed (in Step 3) is
introduced into these host cells. The process inserting rDNA into the host
cell is called transformation. Bacterial cells like E. coli, as well as yeast and
fungi are usually selected as host cells.
 Step 5: Selection of transformed host cells
Those cells that have successfully taken up the rDNA molecules are called
transformed cells (or recombinant cells). In this step, the transformed cells
are separated from the untransformed cells by using various methods that
use marker genes.
 Step 6: Expression and multiplication of the target DNA in the host
It must be ensured that the foreign DNA inserted into the vector DNA
expresses the desired character in the host cells. The transformed host cells
must also be able to be multiplied to obtain sufficient number of copies. If
needed, such genes may also be transferred and expressed into another
organism.

Recombinant DNA technology
process

Polymerase Chain Reaction
(PCR)
 Developed in 1983 by American biochemist Kary Mullis.

 Is used in molecular biology to make many copies of
(amplify) small sections of DNA or a gene.
 Enables the generation of thousands to millions of copies of
a particular section of DNA from a very small amount of
DNA.
 Common tool used in medical and biological research.
 Used in the early stages of processing DNA for sequencing,
for detecting the presence or absence of a gene to help
identify pathogens during infection and when generating
forensic DNA profiles form tiny samples of DNA.

Polymerase Chain Reaction
(PCR)
 5 core “ingredients”:
1. DNA template to be copied
2. Primers
3. dNTPs (DNA nucleotide bases)
4. Taq polymerase enzyme
5. Buffer to ensure the right conditions for the reaction
 3 main stages:
1. Denaturing – double-stranded template DNA is heated to separate
it into 2 single strands
2. Annealing – temperature is lowered to enable the DNA primers to
attach to the template DNA
3. Extending – temperature is raised and the new strand of DNA is
made by the Taq polymerase enzyme.

PCR process

Ethical issues in genetic
engineering
 Transgenics and genetically modified foods

 Health risks, long-term effects on environment.
 Should research be limited and how to decide on the limits?
 Should certain research be banned or morally impermissible
(e.g. research for purely offensive military purposes)?
 Are there fundamental issues with creating new species?
 Are chimeras and transgenics more like to suffer than
“traditional” organisms?
 Will these interventions redefine what it means to be “normal”?

Ethical issues in genetic
engineering (cont’d)
 Social concerns, e.g.:

• If chimeric entities possessing degrees of intelligence are
created, should these entities be given rights and special
protection?
• What, if any, social and legal controls should be placed on such
research?
• What unintended personal, social or cultural consequences
could result?
• Who will have access to these technologies and how will scarce
resources (e.g. medical advances and novel treatments) be
allocated?

Let’s Check!
1. Recombinant DNA technology involves

________ and _______ DNAs.
2. What are the tools used in recombinant DNA
technology?
3. What are the stages involved in the basic
procedure of recombinant DNA technology?
4. What makes Ti plasmid a good cloning vector
for plants?


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Objectives:

 Compare classical breeding with modern

 Outline the steps involved in genetic

SHS General Biology 2 All Rights Reserved

 Mendelian genetics and modern genetics are really parts of the

SHS General Biology 2 All Rights Reserved

 Can be used to produce plants that have a

SHS General Biology 2 All Rights Reserved

 Classical breeding or selective breeding is a form of genetic

SHS General Biology 2 All Rights Reserved

 Technique used in genetic engineering that

SHS General Biology 2 All Rights Reserved

SHS General Biology 2 All Rights Reserved

 Step 1: Select and isolate of target DNA (aka “gene of interest”)

SHS General Biology 2 All Rights Reserved

SHS General Biology 2 All Rights Reserved

SHS General Biology 2 All Rights Reserved

 Developed in 1983 by American biochemist Kary Mullis.

SHS General Biology 2 All Rights Reserved

SHS General Biology 2 All Rights Reserved

SHS General Biology 2 All Rights Reserved

 Transgenics and genetically modified foods

SHS General Biology 2 All Rights Reserved

 Social concerns, e.g.:

SHS General Biology 2 All Rights Reserved

1. Recombinant DNA technology involves

SHS General Biology 2 All Rights Reserved

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