CHAPTER 4

DNA CLONING

MISS NUR SHALENA SOFIAN

INTRODUCTION• Scientists in early years were able to isolate

fragments of DNA in vitro and covalently joined the DNA from different sources – recombinant DNA molecules

• Introduce recombinant DNA into living cells, able to replicate in host cell producing repetitive identical copies – gene cloning

• Application of recombinant DNA technology in diverse areas e.g. gene therapy, screening human diseases, recombinant vaccines, production of transgenic plant and animals

Cloning experiments usually involve two kinds of DNA molecules Chromosomal DNA or cDNA

Serves as the source of the DNA segment of interest

Preparation involves breaking the cells, extract the DNA and purify it via biochemical techniques e.g. chromatography, centrifugation

Vector DNA Serves as the carrier of the DNA segment that is

to be cloned

Cloning Requires DNA Sources

The cell that harbors the vector is called the host cell When a vector is replicated inside a host cell,

the DNA that it carries is also replicated

The vectors commonly used in gene cloning were originally derived from two natural sources 1. Plasmids 2. Viruses

CLONING VECTOR: PLASMID

CHARACTERISTICS

Small circular pieces of ds DNA Self replicating Stably inherited in extrachromosomal state Do not change in size Able to replicate independently of the chromosome Size range 1-200 kb Replication is coupled to host replication in a:

Stringent manner – one or two plasmids made Relaxed manner – 10-200 copies of plasmid made

Carry genes confer for antibiotic resistance: R-factor plasmid

Unique RE sites Promoters for gene expression Contains origin of replication site. Can determine:

a. Which host cells to be used upon replication e.g. if E. coli is chosen as host cell, therefore the origin of replication in vector must be able to be recognized by the host cell

b. Copy number of plasmid

- Stronger origins can achieve higher copy numbers

- lower copy numbers

Commercially available plasmids have selectable markers. Typically, genes conferring antibiotic resistance to the host cell

E.g. plasmid pBR322 contains gene ampr and tetr. Bacteria harboring these antibiotic resistant genes can grow on media containing ampicillin and tetracycline. Bacteria that do not contain the plasmid, is unable to grow because of antibiotic sensitive

E.g. pUC plasmids having multiple cloning site (MCS) where they confer genes for ampr , origin of replication and lacZ (expressing β- galactosidase)

pBR322 plasmid

pUC plasmid

CLONING VECTOR: VIRUS• Virus infecting bacteria cells and propagate themselves

by taking control of the host cell’s metabolic activity – bacteriophage

• Insertion of DNA fragments into viral genome will allow the gene to be replicated along with the viral DNA

• The nucleic acid can be either DNA or RNA• Successful transduction is observed by formation of

viral plaque on media• Utilizing viral vectors are more efficient compared to

plasmid transformation because it yields higher amount of clones

Bacteriophage λ vector

• Promote specialized transduction – important criteria for λ phage

• Λ can be maade to accept insertion of DNA from any sources (in vitro)

• E.g. virus E. coli– linear duplex molecule when isolated from phage– 48.5 kb– No universal λ vectors – its utilization depend on the

objectives of the cloning experiments• Choice of vector:

• Size of DNA fragment to be inserted• RE to be used• Necessity for expression of the cloned genes• Method of screening to be used to select the desired clones

Bacteriophage λ vector

Some Vectors Used in Cloning ExperimentsExample Type Description

pBluescript Plasmid To clone small segments of DNA and propagate them in E. coli

YEP24 Plasmid Known as shuttle vector where it can replicates in 2 different hosts, E. coli and Saccharomyces cerevisiae. It contains origin of replication for both bacteria

λgt11 Viral Contains promoter from lac operon and expressed in E. coli. Also known as expression vector because it is designed to clone coding sequences of genes for successful transcription and translation

SV40 Viral Infects mammalian cells

Baculovirus Viral Infects insect cells and ofter expressed the proteins in large amount

Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments

The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes These bind to specific DNA sequences and then

cleave the DNA at two defined locations, one on each strand

Cloning Requires Enzymes that Cut and Paste DNA

• Restriction enzymes are made naturally by many species of bacteria They protect bacterial cells from invasion by

foreign DNA, particularly that of bacteriophage• RE digest DNA producing either sticky ends or

blunt ends- Sticky ends - ss regions of DNA that hang on one end and can complement sequence of DNA from other sources- Blunt/ cohesive ends – RE cuts in the middle of DNA fragments; no sticky ends

• Restriction enzymes bind to specific DNA sequences These are typically palindromic

For example, the EcoRI recognition sequence is:

5’ GAATTC 3’3’ CTTAAG 5’

• DNA ligase covalently bind the strands together in sugar-phosphate backbone to make it more stable

Question to ponder…If RE can cut up segments of DNA, why do they not destroy the

host’s cell own DNA???

• Almost all RE works with methylases that recognize and methylate the same DNA site

• Combination of RE and methylase – restriction modification system

• After methylation, DNA sites are protected against most RE so DNA remains unharmed in host cell

Naming Restriction Enzymes

• 1st three letters are from the microorganism that produces them

• 1st letter is genus; the next two letters are species e.g. Haemophilus influenza yields Hin

• Strain designation is sometimes included e.g. the ‘d’ from Rd

• The Roman numbering signifies how many of the enzymes produced e.g I, II, III

• E.g HindIII recognizes AAGCTT

This interaction is not stable because it involves only a few hydrogen bonds

Add DNA ligase which covalently links the DNA backbones

To establish a permanent connection, the sugar-phosphate backbones of the two DNA

fragments must be covalently linked

A recombinant DNA molecule

The role of restriction enzyme, EcoRI, in producing recombinant DNA molecule

A closer look… BamHI

5’ … ACTGTACGGATCCGCTA …3’3’ … TGACATGCCTAGGCGAT …5’

PRODUCING…

5’ … ACTGTACG GATCCGCTA …3’3’ … TGACATGCCTAG GCGAT …5’

BamHI

3’ STICKY ENDSCan bind with other DNA

fragment having the same overhangs

5’ … ACTGTACAGATCTGCTA …3’3’ … TGACATGTCTAGACGAT …5’

PRODUCING…

5’ … ACTGTACA GATCTGCTA …3’3’ … TGACATGTCTAG ACGAT …5’

BglII

A closer look… BglII

At the end… the compatible ends meet

5’ … ACTGTACG GATCCGCTA …3’3’ … TGACATGCCTAG GCGAT …5’

5’ … ACTGTACA GATCTGCTA …3’3’ … TGACATGTCTAG ACGAT …5’PRODUCING…

5’ … ACTGTACAGATCCGCTA …3’3’ … TGACATGTCTAGGCGAT …5’

DNA LIGASE

Ligations

• When DNA moleculaes with sticky ends come together, only hydrogen bonds between complimentary nucleotides are reformed

• H-bonds are not stable enough to be permanent

• DNA ligase helps to join the ends of DNA and re-establishes phosphodiester bond in the DNA molecule

A closer look… EcoRV

5’ … ACTGTACGATATCGCTA …3’3’ … TGACATGCTATAGCGAT …5’

PRODUCING…

5’ … ACTGTACGAT ATCGCTA …3’3’ … TGACATGCTA TAGCGAT …5’

BLUNT ENDSCan bind with other DNA molecules with

blunt ends

Host Cell Types

• Depending on the purpose of cloning• E.g. isolate genes – simpler system

expression – require specific system

• Common – simple primary host to isolate sequence introduced into more complex system for expression

• Divided into prokaryotic and eukaryotic host

Prokaryotic Host

• Ideal host cell:– Easy to handle– Available in wide variety of genetically defined

strain– Accept range of vectors– E.g. E. coli

• Should be simple with newly synthesized mRNA immediately available for translation

Eukaryotic Host

• Range from microbes to complex multicellular organisms

• Higher eukaryotes – require specialized solutions

• Commonly used hosts – yeast & fungie.g. S. cerevisiae, A. nidulans, Neurospora crassa

• Plants and animal hosts – grown as cell culture before manipulation

Types of host cell used for GE???

Delivery Methods

• Delivering manipulated vector and insert DNA into host cell – crucial factor determining the success of cloning

• Common delivery methods:a. Transformationb. Transfectionc. Packaging phage DNA in vitro

a. Transformation • Unidirectional transfer of extracellular DNA into

cells• Recipients whose phenotypes are changed by

transformation – transformants• Transformation is enhanced by treating the cells

with chemicals / electroporation – increase cell competency (cell membrane is permeable for exogenous DNA uptake)

• Types of transformation:– Natural - bacteria’s ability to take up DNA naturally– Engineered – bacteria is GE to enable them take up

DNA

b. Transfection

• Transformation of eukaryotic cells by incorporating foreign DNA into cells

• Three basic strategies:1. Stealth – based on the use of positively charged

carrier molecules mixed with experimental DNA (in vitro) and apply directly to cell culture mediae.g. calcium phosphate ppt

2. Attack – using physical methods to force exprimental DNA into cellse.g. biolistic, electrophoration

3. Infection – use recombinant eukaryotic viruses to deliver DNA into host cells

Biolistic shot-gun method

Electrophoration

The use of calcium phosphate precipitation

c. Packaging phage DNA in vitro

• In vitro packaging usually gives higher transduction frequency

• Mix together with phage DNA, head protein + tail protein + packaging enzymes = complete phage particles

• Artificially produced phages are then infect host cells

• The reaction is efficiently performed in very high concentrated mixed lysate – genetic complementation occurs and DNA is packaged