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Gene cloning and cloning vectors

Presented by:Syed KashifDepartment of PharmacologyAACP

2 DNA CLONING

Cloning is the process of producing similar populations of genetically identical individuals that occurs in nature.

Cloning refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms.

DNA cloning is a technique for reproducing DNA fragments

It can be achieved by two different approaches: 

▪ cell based

  ▪ using polymerase chain reaction (PCR). 

A vector is required to carry the DNA fragment of interest into the host cell.  

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DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount.

This technique is the first stage of most of the genetic engineering experiments:

▪ production of DNA libraries

▪ PCR

▪ DNA sequencing 

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Gene of interest is cut out with RE

Host plasmid is cut with same RE

Gene is inserted into plasmid and ligated with ligase

New plasmid inserted into bacterium (transform)

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Enzymes used in molecular cloning

6 Nucleases - Nucleases are a group of enzymes which cleave or cut the genetic

material (DNA or RNA). DNase and RNase Nucleases are further classified into two types based

upon the substrate on which they act. Nucleases which act on or cut the DNA are classified as DNases, whereas those which act on the RNA are called as RNases.

DNases are further classified into two types based upon the position where they act. DNases that act on the ends or terminal regions of DNA are called as exonucleases and those that act at a non-specific region in the centre of the DNA are called as endonucleases.

7 Restriction Enzymes

DNAses which act on specific positions or sequences on the DNA are called as restriction endonucleases.

The sequences which are recognized by the restriction endonucleases or restriction enzymes (RE) are called as recognition sequences or restriction sites. These sequences are palindromic sequences.

Different restriction enzymes present in different bacteria can recognize different or same restriction sites. But they will cut at two different points within the restriction site. Such restriction enzymes are called as isoschizomers.

8 Mode of action The restriction enzyme binds to the recognition site and checks for the

methylation (presence of methyl group on the DNA at a specific nucleotide).

If there is methylation in the recognition sequence, then, it just falls off the DNA and does not cut. If only one strand in the DNA molecule is methylated in the recognition sequence and the other strand is not methylated, then RE (only type I and type III) will methylate the other strand at the required position. The methyl group is taken by the RE from S-adenosyl methionine by using modification site present in the restriction enzymes.

However, type II restriction enzymes take the help of another enzyme called methylase, and methylate the DNA. Then RE clears the DNA. If there is no methylation on both the strands of DNA, then RE cleaves the DNA

9 Types

The restriction endonucleases can be divided into three groups as type I, II and III.

10 Type I Restriction Enzymes

These restriction enzymes recognize the recognition site, but cleave the DNA somewhere between 400 base pairs (bp) to 10,000 bp or 10 kbp right or left.

The cleavage site is not specific. These enzymes are made up of three peptides with multiple functions. These enzymes require Mg++, ATP and S adenosyl methionine for cleavage or for enzymatic hydrolysis of DNA.

These enzymes are studied for general interest rather than as useful tools for genetic engineering.

11 Type II Restriction Enzymes

Restriction enzymes of this type recognize the restriction site and cleave the DNA withinthe recognition site or sequence.

These enzymes require Mg++ as cofactor for cleavage activity and can generate 5 -PO4 or 3 -OH. Enzymes of this type are highly important because of their specificity.

Type II restriction enzymes are further divided into two types based upon their mode of cutting.

12 Blend End Cutters & Cohesive End Cutters Type II Restriction Enzymes - Blunt end cutters Type II restriction enzymes

of this class cut the DNA strands at same points on both the strands of DNA within the recognition sequence. The DNA strands generated are completely base paired. Such fragments are called as blunt ended or flush ended fragments.

Type II Restriction Enzymes - Cohesive end cutter Type II restriction enzymes of this class cut the DNA stands at different points on both the strands of DNA within the recognition sequence. They generate a short single-stranded sequence at the end. This short single strand sequence is called as sticky or cohesive end. This cohesive end may contain 5 -PO4 or 3 -OH, based upon the terminal molecule (5 -PO4 or 3 -OH). These enzymes are further classified as 5end cutter (if 5 -PO 4 is present) or 3 -end cutter (if3' -OH is present).

13 Type III Restriction Enzymes

Type III Restriction enzymes of this type recognize the recognition site, but cut the DNA 1 kbp away from the restriction site. These enzymes are made up of two peptides or subunits. These enzymes require A TP, Mg++ and S-adenosyl methionine for action.

14 Property Type I Type II Type IIIStructure Enzyme complex

of 500600 k dal composed of three separate subunits

Normally homodimers of 20-70 k dal

Heterodimers with subunits of 70 and 100 k dal

Composition Multienzyme complex with R (endonuclease), M (methylase) and S (specificity) subunits

Separate enzymes; endonuclease is a homodimer, methylase a monomer

M subunit provides specificity on its own; functions as methylase; as heterodimer with R subunit; functions as methylase- endonuclease

Cofactors Mg2+, ATP, Sadenosylmethionine (SAM) (needed for cleavage as well as methylation)

Mg2+, SAM (for methylation only)

Mg2+, ATP (for cleavage), SAM (needed for methylation: stimulate cleavage)

15Property Type I Type II Type IIIRecognition sites Asymmetric,

bipartite, may be degenerate; 1315 base pairs containing interruption of 6 to 8 base pairs

Asymmetric, may be bipartite, may be degenerate; 4 to 8 base pairs normally 180° rotational symmetry

Asymmetric, uninterrupted, 5-6 nucleotide long with no rotational symmetry

Cleavage Non-specific, variable distance (100-1000 nucleotides) from recognition site

Precise cleavage within recognition site at defined distance

Precise cleavage at a fixed distance; 25-27 nucleotides from recognition site

Example EcoK EcoRI EcoP1

16 DNA Ligase Recombinant DNA experiments require the joining of two different DNA

segments or fragments in vitro. The cohesive ends generated by some RE will anneal themselves by

forming hydrogen bonds. But the segments annealed thus are weak and do not withstand experimental conditions.

To get a stable joining, the DNA should be joined by using an enzyme called ligase.

DNA ligase joins the DNA molecule covalently by catalysing the formation of phosphodiester bonds between adjacent nucleotides.

17 DNA ligase isolated from E. coli and T 4 bacteriophage is widely used. These ligases more or less catalyse the reaction in the same way and

differ only in requirements of cofactor. T4 ligase requires ATP as cofactor and E. coli ligase requires NADP as

cofactor. The cofactor is first split (ATP - AMP + 2Pi) and then AMP binds to the

enzyme to form the enzyme-AMP complex. This complex then binds to the nick or breaks (with 5' -PO4 and 3' -OH)

and makes a covalent bond in the phosphodiester chain. The ligase reaction is carried out at 400 C for better results.

18 Kinases

Kinase is the group of enzymes, which adds a free pyrophosphate (PO4) to a wide variety of substrates like proteins, DNA and RNA.

It uses ATP as cofactor and adds a phosphate by breaking the ATP into ADP and pyrophosphate.

It is widely used in molecular biology and genetic engineering to add radiolabelled phosphates.

19 Alkaline phosphatases Phosphatases are a group of enzymes which remove a phosphate from a

variety of substrates like DNA, RNA and proteins. Phosphatases which act in basic buffers with pH 8 or 9 are called as

alkaline phosphatases. Most commonly bacterial alkaline phosphatases (BAP), calf intestine

alkaline phosphatases (CIAP) and shrimp alkaline phosphatases are used in molecular cloning experiments.

The PO4 from the substrate is removed by forming phosphorylated serine intermediate. Alkaline phosphatase is metalloenzymes and has Zn++ ions in them.

20 Reverse Transcriptase This enzyme uses an RNA molecule as template and synthesizes a DNA

strand complementary to the RNA molecule. These enzymes are used to synthesize the DNA from RNA. These enzymes are present in most of the RNA tumour viruses and

retroviruses. Reverse transcriptase enzyme is also called as RNA dependent DNA

polymerase. Reverse transcriptase enzyme, after synthesizing the complementary

strand at the 3 end of the DNA strand, adds a small extra nucleotide stretch without complementary sequence. This short stretch is called as R-loop.

GENERATING DNA FRAGMENTS

MECHANICAL SHEARING RESTRICTION DIGEST CDNA SYNTHESIS HYBRIDIZATION METHODS DIRECT CHEMICAL SYNTHESIS

MECHANICAL SHEARING : 1. Random fragments of source DNA can be obtained by

mechanical shearing of bacterial, plant or animal cells.2. Mechanical shearing caused by high speed mixing at

1500 rpm for 30 min. This gives fragments of 8 kb mean size

3. Short single stranded regions – termini or blunt end fragments are formed

4. Sonication can reduce the length of fragment to about 300 nucleotide pairs.

5. Shearing does not necessarily produce 5’ phosphate and 3’ OH ends. Therefore the end of the fragments must be repaired

RESTRICTION ENDONUCLEASE DIGESTION :

1. Large number of restriction enzymes which recognize and cut DNA within target sites of 4 or 5 or 6 or 7 nucleotides are known.

2. Depending upon the number of target sites present, DNA may be cut into too small or too big size fragments.

3. Generally the same restriction enzymes is used for vector and the DNA of interest.

4. The digestion carried out may be light, moderate or heavy producing from small number to large number of fragments which are reproducible

24 PLASMID CLONING STRATEGY

It involves five steps

I. Enzyme restriction digest of DNA sample.

II. Enzyme restriction digest of DNA plasmid vector.

III. Ligation of DNA sample products and plasmid vector.

IV. Transformation with the ligation products.

V. Growth on agar plates with selection for antibiotic resistance.

25 STEP 1. RE DIGESTION OF DNA SAMPLE

26 STEP 2. RE DIGESTION OF PLASMID DNA

27 STEP 3. LIGATION OF DNA SAMPLE AND PLASMID DNA

28 STEP 4. TRANSFORMATION OF LIGATION PRODUCTS

The process of transferring exogenous DNA into cells is call “transformation”

There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells.

A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

29 CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

30 TRANSFORMATION BY ELECTROPORATION

31 STEP 5. GROWTH ON AGAR PLATES

32 Blue colonies represent Ampicillin-resistant bacteria that contain pVector and express a functional alpha fragment from an intact LacZ alpha coding sequence.

White colonies represent Ampicillin-resistant bacteria that contain pInsert and do not produce LacZ alpha fragment

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36 CLONING VECTORS

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube.

Cloning vectors share four common properties:

1. It should be able to replicate autonomously.

2. Contain a genetic marker (usually dominant) for selection.

3. Unique restriction sites to facilitate cloning of insert DNA.

4. Minimum amount of nonessential

37 Types

Plasmid as vector .

Bacteriophage as vector.

Cosmid as vector

Phagemid as vector.

Bacterial Artificial Chromosomes(BACs)

Yeast Artificial Chromosomes(YACs)

Retroviral vectors

38 Plasmid

Bacterial cells may contain extra-chromosomal DNA called plasmids.

Plasmids are usually represented by small, circular DNA. Some plasmids are present in multiple copies in the cell

39 Plasmid vectors are ≈1.2–3kb and contain:

replication origin (ORI) sequence

a gene that permits selection,

Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.

Exogenous DNA can be inserted into the bracketed region .

40 SELECTIVE MARKER Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker the plasmid becomes useful for the

cell. Under the selective conditions, only cells that contain plasmids with selectable

marker can survive Genes that confer resistance to various antibiotics are used. Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

41 ORIGIN OF REPLICATION

Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes.

Without replication origin, DNA cannot be replicated in the cell.

42 MULTIPLE CLONING SITE Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment

with several unique sites for restriction endo- nucleases located next to each other

Restriction sites of the polylinker are not present anywhere else in the plasmid.

Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

EcoRI – Escherichia coli strain RBamHI – Bacillus amyloliquefaciens DpnI – Diplococcus pneumoniae, HindIII – Haemophilus influenzae, BglII – Bacillus globigii,PstI – Providencia stuartii 164,Sau3AI – Staphylococcus aureus 3A,KpnI – Klebsiella pneumoniae, 1st

43Plasmid Vector: pBR322

Derived from E. coli plasmid ColE1), which is 4,362 bp DNA and was derived by several alterations in earlier cloning vectors.

pBR322 is named after Bolivar and Rodriguez, who prepared this vector.

It has genes for resistance against two antibiotics (tetracycline and ampicillin), an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific restriction enzyme.

It has unique restriction sites for 20 restriction endonucleases. Certain restriction sites for eg., BAM HI in the tetr genes of the

plasmid are present within the gene in such a way that the insertion of foreign segment of DNA will inactivate the tetr gene.

The recombinant plasmid will allow the cells to grow only in the presence of ampicillin but will not protect them against tetracyclin.

Thus recombinant plasmids selection will be easily carried out.

44 Plasmid Vector: pBR322

• Contains: • Selectable Markers:

Ampicillin resistance gene. Tetracycline resistance gene. Col E I replication origin. Eco RI site.

45 Plasmid Vector: pUC vectors Another series of plasmids that are used as cloning vectors belong to pUC series (after the place

of their initial preparation I.e. University of California). These plasmids are 2,700 bp long and possess Ampicillin resistance gene The origin of replication derived from pBR322 and The lacz gene derived from E.coli. Within the lac region is also found a polylinker sequence having unique restriction sites. When DNA fragments are cloned in this region of pUC, the lac gene is inactivated. These plasmids when transformed into an appropriate E. coli strain, which is lac (JM103, JM109),

and grown in the presence of IPTG (isopropyl thiogalactosidase, which behaves like lactose, and induced the synthesis of b-galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.

46 On the other hand, pUC having no inserts are transformed into bacteria, it will

have active lac Z gene and therefore will produce blue colonies, thus permitting identification of colonies having pUC vector with cloned DNA segments.

The cloning vectors belonging to pUC family are available in pairs with reverse orders of restriction sites relative to lac Z promoter. pUC8 and pUC9 are one such pairs.

Other similar pairs include

pUC12 and pUC13 and

pUC18 and pUC19.

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Hind II

PstI Sal IBamHI

Sma IEcoRI

EcoRI

Sma IBamHI Sal I

PstIHind II

Lac ZOperator

Promoter

B-galactosidase gene

Ampicillin resistance

pUC Hae II

Hae II

48 Plasmid Vector: pUC vectors

Contains

Ampicillin resistance gene.

Multiple cloning site.

ColEI ( origin ).

49 Bacteriophage lambda ( )λ Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as

host. Its structure is that of a typical phage: head, tail, tail fibres.

The bacteriophages used for cloning are the phage  and M13 phage.λ It infects bacteria.

Follow either lytic or lysogenic cycle.

There are two kinds of phage vectors –λ insertion vector and replacement vector.

Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted.

In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.

Size is 48,502 bp.

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PHAGE M13 VECTORBacteriophage lambda

COS site: Cohesive “sticky” ends

Lysis

Lysogeny

Head

Tail

Replication

Circularized lambda

ori

52 Cosmids A cosmid is a type of hybrid plasmid that contains a Lambda

phage cos sequence.  Cosmids are plasmids that incorporate a segment of bacteriophage λ

DNA that has the cohesive end site (cos) which contains elements required for packaging DNA into λ particles. It is normally used to clone large DNA fragments between 28 to 45 Kb.

Cosmid can replicate in bacterial cell, so infected cells grow into normal colonies

Insert DNA limited by the amount of DNA that can fit into phage capsule

Somewhat unstable, difficult to maintain

cos

TetR

EcoRI

21.5 kbori

Cos site is the only requirement for packaging into phage particle

53 Latest Generation VECTOR: Phagemid- pBluescript

A phagemid or phasmid is a plasmid that contains an f1 origin of replication from a f1 phage.

It can be used as a type of cloning vector in combination with filamentous phage M13. 

Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles.

Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques, such as transformation and electroporation.

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55 Bacterial Artificial Chromosomes(BACs)

BACs can hold up to 300 kbs.

The F factor of E.coli is capable of handling large segments of DNA.

Recombinant BACs are introduced into E.coli by electroportation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor.

Example: pBAC108L

Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.

A chloramphenicol resistance gene, and a cloning segment.

56 Yeast Artificial Chromosomes

Purpose:Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic ChromosomesClone very large inserts of DNA: 100 kb - 10 MbFeatures:YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome

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Additional features:

Often have a selection for an insert

YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a selection marker for propogation of the YAC through bacteria.

The YAC can use both yeast and bacteria as a host

58 RETROVIRAL VECTORS

Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells.

Retroviruses are RNA viruses. The viral RNA is converted into DNA by the viral reverse transcriptase and then is

efficiently integrated into the host genome Any foreign or mutated host gene introduced into the retroviral genome will be

integrated into the host chromosome and can reside there practically indefinitely. Retroviral vectors are widely used to study oncogenes and other human genes.

APPLICATIONS OF GENE CLONING IN RESEARCH

1. Identifying the genes in a genome sequence2. Determining the function of an unknown gene3. To study the transcriptome and proteomeI. The transcriptome, which is the messenger RNA

(mRNA) content of a cell, and which reflects the overall pattern of gene expression in that cell.

II. The proteome, which is the protein content of a cell and which reflects its biochemical capability.

4. Studying protein–protein interactions

APPLICATIONS OF GENE CLONING IN BIOTECHNOLOGY

Production of Protein from Cloned Genes Production of recombinant pharmaceuticals in

medicine. Eg. Recombinant insulin Synthesis of human growth hormones in E. coli Recombinant vaccines eg. Vaccine for hepatitis B The gene addition approach to plant genetic

Engineering in agiculture field eg. Plants that make their own insecticides, Herbicide resistant crops

REFERENCE

Gene cloning by T.A.BROWN Molecular Biology Of The Cell By

ALBERTS INTERNET source

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