Chapter 8.0 RECOMBINANT DNA TECHNOLOGY · Cloning Vectors: Cosmid • Plasmid with yeast chromosome...

Chapter 8.0

RECOMBINANT DNA TECHNOLOGY

OVERVIEW

RECOMBINANT

DNA

TECHNOLOGY

METHODS

IN GENE

CLONING

APPLICATION OF

RECOMBINANT DNA

TECHNOLOGY

• 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)

(c)Explain the characteristics of plasmid as cloning and expression vector

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

(e)Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation

• (ii) Taq polymerase for DNA amplification using PCR.

Learning

Outcomes

Definition of Recombinant DNA Technology

• Formation of new combinations of genes by

isolating genes from the organism and introducing

them into either a similar or unrelated organism.

PURPOSES Enable scientists to obtain many copies of specific DNA segment for the purpose of

studying it

Modifying the DNA of an organism to produce new

genes with new traits.

Tools Used

in Cloning

Target DNA

Host Cell

Modifying

Enzymes Restriction

Enzyme

DNA Cloning Vector

Target DNA

A selected DNA that contain gene of interest.

Restriction enzymes

A molecular scissors that cut the single strand

and the double strand at specific point

Examples: EcoR1 to produce sticky ends Sma1 to produce blunt ends

DNA CLONING VECTOR

A plasmid of bacteria that brings the foreign DNA fragment

into the genome of the host cell

Examples: Plasmid, Bacteriphage, Cosmid, Yeast Artificial Chromosomes

HOST CELL

A cell that receives recombinant DNA

for cloning purpose

HOST CELL

Enzymes used in join targeted DNA fragment with

the vector to form recombinant DNA.

MODIFYING ENZYME

Example: DNA Ligase, Taq polymerase

Restriction endonucleases.

• Extracted from bacteria.

• Naturally used to cut viral DNA into small

fragments at specific base/site.

– For defense.

Restriction Enzymes

RESTRICTION ENZYME

Enzymes Source

EcoRI Escherichia coli

BamHI Bacillus amyloliquefaciens

SmaI Serratia marcescens

• Splice through DNA at specific base sequence.

• Specific base sequence: Restriction sites.

• Most of the base are palindromic.

Restriction sites

• Palindromic?

Base sequence of one strand reads the same as its complement strand in opposite direction.

Enzymes Restriction Sites

EcoRI 5’-GAATTC-3’ 3’-CTTAAG-5’

BamHI 5’-GGATCC-3’ 3’-CCTAGG-5’

SmaI 5’-GGGCCC-3’ 3’-CCCGGG-5’

Example: EcoRI

• Most make staggered cut in two strands forming sticky ends.

breaking the phosphodiester bond

RESTRICTION ENZYME THAT PRODUCE STICKY ENDS:

• Example: SmaI

• Some cut straight across both strand forming blunt ends.

RESTRICTION ENZYME THAT PRODUCE BLUNT ENDS:

DNA cloning vector

• A small piece of DNA which a foreign DNA fragment can be inserted.

• Example: Plasmid

Bacteriophage

Cosmid

Yeast artificial chromosomes (YACs)

Types

DNA Inserts Capacity

(1 kb = 1000 nucleotides)

Form of vector Example

Plasmid 10 kb Double stranded

circular DNA pUC18

Bacteriophage 20 kb Linear DNA λ2001

Cosmid 35-45 kb Double stranded

circular DNA sCOS-1

YACs – Yeast Artificial Chromosomes

200-1500 kb Double stranded

circular DNA

pYAC

Cloning Vectors: Plasmid

21

• Small ring-shape DNA

in bacteria.

• Not part of

chromosome.

• Self-replicating.

• Has small number of

genes.

• Example: pUC18

Cloning

• Viruses that infect bacteria

• Can carry larger DNA

inserts than bacterial plasmid

• Eg. λ2001

Cloning Vectors: Bacteriophage

• Hybrids of plasmid

and bacteriophage lambda DNA.

• Can accommodate large inserts of DNA

• eg.: Scos-1

Cloning Vectors: Cosmid

• Plasmid with yeast chromosome

• Eg. pYAC – capacity

is bigger than other vectors

Cloning Vectors: YACs

Cloning vector: Characteristics

1. Able to accept

foreign DNA in

multiple cloning sites (MCS).

Example: Plasmid

Multiple Cloning

Sites

2. Able to replicate freely in host cell.

Present of origin of

replication initiation -ori gene

Cloning vector: Characteristics

3.Possess selectable genetic marker

a. resistance to antibiotic

eg: ampR, tetR,

kanR

b. lacZ gene

encode for β-

galactosidase

Cloning vector: Characteristics

Host Cell

A cell that can be utilized for DNA cloning in order

to accept, maintain and allow the reproduction of

cloning vector.

Example: Bacteria

1. Able to receive DNA recombinant through the transformation process.

Host cell: Characteristics

2. Able to maintain the structure of DNA recombinant from one generation to other.

Host cell: Characteristics

3. Able to amplify the gene product from the DNA recombinant.

gene product e.g insulin

Host cell: Characteristics

Modifying Enzymes

• Enzymes used in modification of DNA.

Example: DNA Ligase

✓Catalyzed the formation of phosphodiester bonds between adjacent nucleotides in DNA.

• Modifying enzymes: Joins targeted DNA fragment with the vector to form recombinant DNA.

• Examples of modifying enzymes are: DNA ligase and Taq polymerase

NEXT LECTURE

METHODS IN GENE CLONING