II. Restriction EndonucleasesII. Restriction Endonucleases

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

• Restriction Enzymes– Bacterial enzymes that cut DNA molecules

only at restriction sites.– Categorized into two groups based on type

of cut• Cuts with sticky ends• Cuts with blunt ends

A. Origin and functionA. Origin and function• Bacterial origin = enzymes that cleave foreign

DNA • Named after the organism from which they

were derived– EcoRI from Escherichia coli– BamHI from Bacillus amyloliquefaciens

• Protect bacteria from bacteriophage infection– Restricts viral replication

• Bacterium protects it’s own DNA by methylating those specific sequence.

B. AvailabilityB. Availability

• More than 800 types of REs are known; and more than 400 of them are available commercially.

C. ClassesC. Classes

• Type I– Cuts the DNA on both strands but at a non-

specific location at varying distances from the particular sequence that is recognized by the restriction enzyme

– Therefore random/imprecise cuts– Not very useful for rDNA applications

• Type II– Cuts both strands of DNA within the

particular sequence recognized by the restriction enzyme

– Used widely for molecular biology procedures

– DNA sequence = symmetrical

• Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence

• Some enzymes generate “blunt ends” (cut in middle)

• Others generate “sticky ends” (staggered cuts)

– H-bonding possible with complementary tails– DNA ligase covalently links the two fragments

together by forming phosphodiester bonds of the phosphate-sugar backbones

Figure 8.2 Figure 8.2 Actions of restriction enzymes-overviewActions of restriction enzymes-overview

DNA Ligase in Action!DNA Ligase in Action!

III. Vectors for Gene III. Vectors for Gene CloningCloning

•Vectors:– Nucleic acid molecules that deliver a gene

into a cell

– Useful properties:

• Small enough to manipulate in a lab

• Survive inside cells

• Contain recognizable genetic marker

• Ensure genetic expression of gene

• Autonomous replication.

A. Requirements of a vector to serve A. Requirements of a vector to serve as a carrier moleculeas a carrier molecule

• The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently

• Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells.

• Some vectors contain an additional eukaryotic origin of replication allowing autonomous, episomal replication in eukaryotic cells.

• Multiple unique cloning sites are often included for versatility and easier library construction.

CONTD…

B. Main types of vectorsB. Main types of vectors

i. Bacterial Plasmids

ii. Bacteriophages

iii. Cosmids

iv. Others: Bacterial artificial chromosome (BAC), Yeast artificial chromosome (YAC), Human

artificial chromosome (HAC), yeast 2 micron plasmid, retrovirus, baculovirus vector……

C. Choice of vectorC. Choice of vector

• Depends on nature of protocol or experiment

• Type of host cell to accommodate rDNA

– Prokaryotic

– Eukaryotic

D. Plasmid vectorD. Plasmid vector

• Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria

• Many confer drug resistance to bacterial strains• Origin of replication present (ORI)

• Examples– pBR322

• One of the original plasmids used• Two selectable markers (Amp and Tet resistance)• Several unique restriction sites scattered throughout

plasmid (some lie within antibiotic resistance genes = means of screening for inserts)

EE. Common steps . Common steps involved in isolating a involved in isolating a particular DNA fragment from a complex particular DNA fragment from a complex mixture of DNA fragments or moleculesmixture of DNA fragments or molecules

1. DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes

2. These products of digestion are inserted into a DNA molecule called a vector Enables desired fragment to be replicated in cell culture to very high levels in a given cell (copy #)

3. Introduction of recombinant DNA molecule into an appropriate host cell

– Transformation or transfection

– Each cell receiving rDNA = CLONE

– May have thousands of copies of rDNA molecules/cell after DNA replication

– As host cell divides, rDNA partitioned into daughter cells

CONTD…

• 4. Population of cells of a given clone is expanded, and therefore so is the rDNA.– Amplification– DNA can be extracted, purified and used for

molecular analyses• Investigate organization of genes• Structure/function• Activation• Processing

CONTD…

• Gene product encoded by that rDNA can be characterized or modified through mutational experiments

• Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct.

• Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.

CONTD…

• Modern vectors contain multi-functional elements designed to permit a combination of cloning, DNA sequencing, in vitro mutagenesis and transcription and episomal replication.

– pUC18• Derivative of pBR322• Advantages over pBR322:

– Smaller – so can accommodate larger DNA fragments during cloning (5-10kbp)

– Higher copy # per cell (500 per cell = 5-10x more than pBR322)

– Multiple cloning sites clustered in same location = “Polylinker”

• Vectors able to survive under antibiotic selection are amplified in bacterial hosts by autonomous replication

• Plasmid DNA containing the gene of interest is purified from large scale cultures

• Subsequent steps in the experimental design are undertaken:– Sub-cloning– Mutagenesis– Sequencing– Transfection of eukaryotic cell lines (calcium

phosphate precipitation, lipofection, electroporation, dextran sulfate, microinjection,…..)

– Fragment isolation for transgenic mice production (microinjection)

– PCR

E. Lambda vectorE. Lambda vector(Bacteriophages)(Bacteriophages)

• Bacteriophage lambda (λ) infects E. coli• Lambda phage is another choice vector. In view of

the disadvantage in case of plasmids (the plasmids can not carry large fragments of DNA) there is a need of other cloning vectors capable of carrying larger fragments.

• λ- phages were found to be capable of carrying much larger DNA inserts, as large as 40 kb.

• Double-stranded, linear DNA vector – suitable for library construction

• Can accommodate large segments of foreign DNA

F. Cosmid vectorsF. Cosmid vectors

An artificially constructed circular DNA molecule of 5000 – 7000 base pairs.

It combines useful features of both, the plasmid and the lambda-phase, because of the presence of the following elements:

i. An origin of replication.ii.Packaging signals of λ-phage, called cos-sites.

Contd…Contd…

iii. In addition, the cosmid contains an antibiotic resistant gene and several restriction sites where foreign DNA can be inserted.

Note: Cosmid permits insertion of large fragments of DNA, up to 35 – 50kb long.

G. Shuttle vectorsG. Shuttle vectors• Hybrid molecules designed for use in multiple

cell types• Multiple ORIs allow replication in both

prokaryotic and eukaryotic host cells allowing transfer between different cell types– Examples:

• E. coli yeast cells• E. coli human cell lines

• Selectable markers and cloning sites

H. Bacterial artificial H. Bacterial artificial chromosomes (BACs)chromosomes (BACs)

• Based on F factor of bacteria (imp. In conjugation)• Can accommodate 1 Mb of DNA (= 1000kbp)• F factor components for replication and copy #

control are present• Selectable markers and cloning sites available• Other useful features:

• Direct RNA synthesis • RNA probes for hybridization experiments• RNA for in vitro translation

I. Yeast artificial chromosomes I. Yeast artificial chromosomes (YACs)(YACs)

• Hybrid molecule containing components of yeast, protozoa and bacterial plasmids– Yeast:

• ORI = ARS (autonomously replicating sequence)• Selectable markers on each arm (TRP1 and URA3)• Yeast centromere

– Protozoa= Tetrahymena• Telomere sequences (yeast telomeres may also be used)

– Bacterial plasmid• Polylinker

• Can accommodate >1Mb (1000kbp = 106 bp)

J. Human artificial chromosomes J. Human artificial chromosomes (HACs)(HACs)

• Developed in 1997 – synthetic, self-replicating• ~1/10 size of normal chromosome• Micro-chromosome that passes to cells during mitosis• Contains:

– ORI– Centromere– Telomere– Protective cap of repeating DNA sequences at ends

of chromosome (protects from shortening during mitosis)

– Histones provided by host cell

Figure 8.3 Figure 8.3 Producing a recombinant vectorProducing a recombinant vectorAntibioticresistancegene

Restrictionsite

mRNA for humangrowth hormone (HGH)

Reversetranscription

Plasmid (vector)

cDNA for HGH

Restrictionenzyme

Restrictionenzyme

Sticky ends

Gene for humangrowth hormone

Ligase

Recombinant plasmid

Introduce recombinantplasmid into bacteria.

Recombinantplasmid

Bacterialchromosome

Inoculate bacteriaon media containingantibiotic.

Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.