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CHAPTER 8: RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.1 Recombinant DNA Technology

LEARNING OUTCOMES : a) Define recombinant DNA technology. b) Define and explain the tools used in recombinant DNA technology, target

DNA, restriction enzymes, DNA cloning vector, host cell and modifying

enzymes.

(c) Explain restriction enzyme and examples of enzymes that produce sticky ends.

(EcoRI: G AATTC) and blunt ends (SmaI : CCC GGG) (d) Explain the characteristics of plasmid as cloning and expression vector

(e) Explain the characteristic of E.coli as host cell and its characteristics

(f) Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation (ii) Taq polymerase for DNA

amplification using PCR.

.

MAIN IDEAS /KEY POINT

EXPLANATION NOTES

Recombinant

DNA technology

It’s a technology that forms a new combination of DNA when

segments of DNA from two different sources (often different

species) in vitro.

Purpose

• Enable scientists to obtain copies of specific DNA segment

for the purpose of studying it.

• Modifying the DNA of an organism to produces new genes

with new traits.

Tools Used in Cloning

1. Target DNA (Genes of interests).

It’s a fragment of chromosomal to be cloned. An enzyme

must be used to cleave fragments that containing genes of

interests.

2. Restriction Enzymes / Restriction of endonucleases.

This enzyme is extracted from a specific bacteria, for example

E.coli. Naturally it is used to cut/splice viral DNA into small

fragments at specific base sequence/ restriction sites. Most of

the base are palindromic. Palindromic is a base sequence of one

strand reads the same as its complement strand in opposite

direction.

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3. DNA cloning vector

A small pieces of DNA which a foreign DNA fragment can

be inserted. For examples, plasmid, bacteriophage, cosmid

and YACs. Every vector must have these characteristics:

1. Able to accept foreign DNA in multiple cloning sites

(MCS)

2. Able to replicate freely in host cell.

Present of origin of replication initiation -ori gene

3. Possess selectable genetic marker

a. resistance to antibiotic.

eg: ampR, tetR, kanR

b. lacZ gene

encode for B- galactosidase

4. Host Cell

A host cell is a cell that has been introduced

with DNA (or RNA), such as a bacterial cell acting as a host

cell for the DNA isolated from a bacteriophage. This cell is

utilized from the DNA cloning to accept, maintain and allow

the replication of cloning vector. This cell needs to have

these characteristics:

1. Able to receive DNA through the transformation

processes.

2. Able to maintain the structure of DNA recombinant

from one generation to other.

3. Able to amplify the gene product from the DNA

recombinant

5’

C T T A A G

3’

G A A T T C

3’ 5’

amp R

lacZ

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5. Modifying Enzymes

This is an enzyme used in the modification of DNA. For

example, DNA ligase. This enzyme catalyzed the formation of

phosphodiester bond between adjacent nucleotides in DNA.

Another example is, Taq polymerase. This is a DNA

polymerase isolated from thermophilic bacterium called

Thermus aquaticus. It catalyzes the addition of nucleotides to

DNA sequences. This enzyme is able to withstand high

temperature thus, it won’t be denatured.

Restriction

enzyme,

examples of

enzymes that produce sticky

ends.

Each restriction enzyme is very specific, recognizing a

particular short DNA sequence (restriction site), and cutting

both DNA strands at precise points within this restriction site.

(Campbell, 2011). Most restriction enzyme make staggered cut

in two strands, forming sticky end. For example, EcoRI. But

some enzyme could also cut straight across both strand, forming

blunt end. For example, SmaI. The table below shows the

restriction site that specifies the restriction enzyme.

Enzymes Restriction Sites

EcoRI 5’-GAATTC-3’

3’-CTTAAG-5’

BamHI 5’-GGATCC-3’

3’-CCTAGG-5’

SmaI 5’-GGGCCC-3’

3’-CCCGGG-5’

The list and

explanation the types of cloning

vectors.

The original plasmid is called a cloning vector, defined as a

DNA molecule that can carry foreign DNA into a host cell

and replicate there. These are the examples of cloning

vectors.

1) Plasmid

It is a small ring-shape DNA in bacteria (only a small

number of genes). It has the form of double stranded

circular DNA. However, it is not part of the bacteria’s

chromosome. But, it can self – replicating. For example,

pUC18

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Bacterial DNA Plasmid

2) Bacteriophage

Bacteriophage is a virus that infected bacteria. The genetic

information is in a linear form. It carries a larger DNA

inserts capacity then the bacteria. For example, λ2001

3) Cosmid

Cosmids can be used to build genomic libraries. Cosmids can contain 37 to 52 kb of DNA, limits based on the

normal bacteriophage packaging size. They can replicate

as plasmids if they have a suitable origin of replication

(ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, They frequently

also contain a gene for selection such as antibiotic

resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. (Hybrids

of plasmids and bacteriophage lambda DNA). Other

example is SCOS-1

Genetic information

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4) YACs

Yeast artificial chromosomes (YACs) are genetically

engineered chromosomes derived from the DNA of the

yeast, Saccharomyces cerevisiae, which is then ligated into

a bacterial plasmid. By inserting large fragments of DNA,

from 100–1000 kb, the inserted sequences can be cloned

and physically mapped using a process called chromosome

walking. This is the process that was initially used for

the Human Genome Project, however due to stability issues,

YACs were abandoned for the use of Bacterial artificial

chromosomes (BAC). This chromosome is a double

stranded circular DNA. Other words, it is a plasmid with

yeast chromosome.

• Eg. pYAC

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CHAPTER 8: RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.2 Methods in Gene Cloning LEARNING OUTCOMES : a) Overview using diagram to show the steps in gene cloning by using plasmid b) Describe the steps in gene cloning by using plasmid as the vector

(i) isolation of gene

(ii) cleave/cut

(iii) insertion (iv) transformation and amplification

(v) screening (blue/white screening)

MAIN IDEAS /KEY POINT

EXPLANATION NOTES

Cloning

Cloning is the process of producing genetically identical individuals

of an organism either naturally or artificially.

Gene Cloning

Is a process that produce many copies of a gene of interest by

making many identical copies of a gene by inserting the gene into a

living host cell (bacteria). These copies can be used in sequencing

the gene.

• 2 methods to copies the DNA fragment:

a. Cloning

b. Polymerase Chain Reaction (PCR)

Steps in Gene

Cloning (Refer

to Campbell,

2011, (9th Ed.),

Pg. 445)

1. Isolation of gene and vector (plasmid) from source (different

organisms).

• Isolation of plasmid (as a vector) from bacteria.

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• Isolation of target DNA or gene of interest from sources

Eg: Human cells, Plant cells, Animal cells

2. Cleave or cut of DNA/gene of interest and plasmid by using the

same restriction enzyme

• The target DNA and plasmid are cut at palindromic

sequence (restriction site) with the same restriction enzyme.

Single cut within the lacZ gene, caused the plasmid to open

and the disruption of the gene. However, many cuts within

the target DNA produced thousands of fragments with

different sizes.

3. Insertion of gene of interest into plasmid (vector).

• Mix the cut plasmid and DNA fragments allowing base pair

between their complementary sticky ends by forming

hydrogen bonds. DNA ligase is added to seal them together

at the sugar phosphate backbones of the fragments whose

sticky ends have base-paired with phosphodiester bond.

Thus, forming a recombinant DNA.

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• BUT, not all plasmid will join with the target DNA. This

caused two types of recombinant DNA:

Non recombinant plasmid

Recombinant plasmid

4. Transformation of recombinant DNA into host cells (bacteria)

and amplification.

– Host cells take up recombinant DNA after being

introduced to it. BUT, not all host cells will take in

the recombinant DNA. Only a small proportion of

bacteria will be transformed.

• There will be THREE conditions:

1. Transformed with non-recombinant

plasmid

2. Transformed with recombinant

plasmid

3. Not transformed

Then, they are mixed in a medium containing calcium chloride. This

solution caused the bacterial cell wall to become permeable. The

host cells reproduce by binary fission and amplify the target DNA.

Inserted gene of

interest

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5. Screening

The mixed bacteria are plate on nutrient-containing agar

medium supplemented with ampicillin and X-gal, a molecule

resembling lactose.

– Purpose: To identify bacterial colonies that carry

gene of interest / target DNA = Blue-white

screening.

• A plasmid vector contains two genes encoding for :

– Resistance to antibiotic (ampR)

– Enzyme β-galactosidase (lacZ)

–

• When cultured in medium containing ampicilin and X-gal:

Bacteria without plasmid fail to grow because of

none ampR gene – not resistance to antibiotic

• Bacteria with plasmid live but with TWO conditions:

– Contain non-recombinant plasmids.

• lacZ gene that encodes for β-galactosidase is

functioning

• Hydrolyzed X-gal BLUE colonies

– Contain recombinant plasmids.

• lacZ gene that encodes for β-galactosidase is

disrupted.

• X-gal not hydrolyzed WHITE colonies.

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BIOLOGY

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CHAPTER 8 : RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.3 Application of Recombinant DNA Technology LEARNING OUTCOMES a) Briefly explain applications of Recombinant DNA Technology in mass

production of insulin using cDNA. b) Describe the steps in production of insulin using cDNA.

MAIN IDEAS

/KEY POINT EXPLANATION NOTES

Application Of

Recombinant

DNA

Technology

• Many fields benefit from DNA technology and genetic engineering.

- Agriculture

- Forensic

- Medicine – insulin for diabetics

- Environment

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Complementary

DNA (cDNA)

cDNA is DNA molecule made in vitro using mRNA as a template

and the enzyme reverse transcriptase.

A cDNA molecule lack introns compared to DNA of the genome

Can be used directly to express the proteins.

cDNA is often used to clone eukaryotic genes in prokaryotes

cDNA is used because:

cDNA represents the expression of the genes without the

introns.

there is no process for RNA splicing in bacteria cell.

Production of

insulin

1. mRNA for insulin is isolated from the beta cells of islets of

Langerhans in the pancreas.

2. Reverse transcriptase enzyme is added to synthesis a cDNA by

using mRNA as template. mRNA strand then discarded by using

mRNA-degrading enzyme.

3. DNA polymerase enzymes is added and synthesizes a second

DNA strand, complementary to cDNA in vitro.

• cDNA : A DNA molecule made in vitro using mRNAas a template and the enzyme reverse transcriptase.

• A cDNA molecule therefore corresponds to a gene, but lacks the introns present in the DNA of the genome.

• Can be used directly to express the proteins

encoded by them. 47

Complementary DNA ( cDNA )

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4. Restriction enzyme ( BamHI ) is used to cut the cDNA and isolate

just the sequence that encodes for the insulin protein.

5. Plasmid removed from a bacterial cell is cut at a specific site using

the same restriction enzyme (BamHI)

6. The cDNA is inserted to the plasmid using DNA ligase. The

recombinant plasmid carrying the human DNA for insulin.

7. The recombinant DNA then transform into the host(E. coli) by

transformation.

8. The bacterium E. coli with its recombinant plasmids, is allowed

to reproduce and undergo screening process.

9. Finally, a lot of bacterial clone carrying many copies of the gene

for insulin will be produced.