l1 3 introduction dna 2014

7/21/2019 L1 3 Introduction DNA 2014

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PR3104 PHARMACEUTICAL BIOTECHNOLOGYAY 2013/14

Dr Chew Eng Hui (Module Coordinator)Office: S4, #03-05Phone: 6516 1955Email: [email protected]

Dr Rachel EeOffice: S4, #03-04Phone: 6516 2653Email: [email protected]

A/P Victor YuOffice: S4, #03-07Phone: 6516 8216

Email:[email protected]



Scope of Study (RE)

• Basic molecular biotechnologyTools & techniques used when working with DNA.

Tools & techniques needed to clone and identify genes: host cells, vectors, restrictionenzymes etc, Polymerase Chain Reaction, site-directed mutagenesis.

• Physicochemical properties of therapeutic proteinsProtein structure: amino acids, peptide bond, ionization, intermolecular forces, proteinfolding, protein stability, solubility, hydrophobicity.

• Production of therapeutic proteinsDownstream processing: isolation of proteins from cells, purification and identification.Formulation of therapeutic proteins into dosage forms.

• Insulin as a recombinant protein



Reference Materials

• Gene cloning and DNA analysis: an introduction / T.A. Brown QH442.2Bro 2006 RBR or QH442.2 Bro 2010

• Pharmaceutical biotechnology: fundamentals and applications / editedby Daan J.A. Crommelin, Robert D. Sindelar, Bernd Meibohm RS380Pha 2008 RBR

• Biotechnology and Biopharmaceuticals – Transforming Proteins andGenes into Drugs / Rodney J. Ho and Milo Gibaldi Second edition;Full-text online via NUS Libraries



Assessments

• 1 online MCQ self-assessment/ refresher on “Basics of DNAstructure and function”/ Posted on IVLE on Jan 17 / Due Jan 30

• CA1 MCQ and short structured questions / L1-7 / Mar 3

• Tutorial and CA Review / L1-7 / Mar 6



Definition of Biotechnology

“ Using living things to create products or to do tasks for

human beings”

• Biotechnology was termed in 1919 by Karl Ereky - convertingraw materials into a more socially useful product

• Used years ago to produce foods and increase crop yields

Traditional Biotechnology

• Modern biotechnology

Recombinant DNA techniques (rDNA or genetic engineering)

Pre‐Recombinant DNA Era

(Before 1970s)

Post ‐Recombinant DNA Era



Translation of biological molecules into therapeutic products

1940s

1970s

1980s

1990s

2000s

Taken from Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs, 2nd Edition, Rodney J.Y. Ho.

Pre‐

Recombinant

DNA

Era

(Before 1970s)

Post ‐Recombinant DNA Era

The discovery of protein, cell, bacteria and Mendelian genetics in 1830-1900, and theinnovative milestones in modern genetics and molecular engineering, provided the basis for exponential growth in the ability to identify, validate and produce biological molecules for therapeutic applications.



NOVO STORY OF INSULINHTTP://WWW.YOUTUBE.COM/WATCH?V=CZEPQ3KKWHO

An example of biopharmaceutical production in the pre-recombinant-DNA Era



Emergence of Biopharmaceuticals

• Identification of biomolecules (e.g. antibodies, blood products,insulin)

• Widespread use is possible if sufficient quantities are collected

• Produced naturally in exceedingly low amounts

• Chemical synthesis/semi-synthesis not useful for large proteins

• Two discoveries (mid-1970s) overcame such difficulties



Emergence of Biopharmaceuticals

1. Genetic EngineeringFacilitates the large-scale production of virtually any protein once its amino acid

sequence has been determined.

Discovery highlights:• Restriction enzymes

• DNA Polymerases (for sequencing and amplifying DNA)

• Manipulating (cloning and mutagenesis) and propagating DNA usingbacterial plasmids

• PCR Technology

2. Hybridoma TechnologyFacilitates the large-scale production of

monoclonalantibody



Investment by biopharmaceutical companies

Pioneeringbiotech cos.?

Genentech

ChironImmunex

Cetus

Taken from Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs, 2nd Edition, Rodney J.Y. Ho.



Worldwide Market for Biopharmaceuticals

• Global pharmaceutical sales in 2010 - $850 bln

• Biotech drugs/biologics accounted for $140 bln (2010)

• ~12% (2001); 19% (2006); 29% (2011) of world’s pharmaceutical market

• Protein drugs• $33 blns in 2004

• Annual growth rate of 12% from 2003 through 2008

• Affected by financial crisis in 2008, but showing signs of rebound in 2010.

Source: IMS Health, Nature Biotechnology 29, 585-591 (2011)

Growth trends in US biotechmarket for biologics (2007-2011)



Nature Biotechnology 30, 1191-1197 (2012)

T 25 bi t h d b d ld id l



Top 25 biotech drugs based on worldwide sales

Taken fromBiotechnology and

Biopharmaceutical

s: Transforming

Proteins and

Genes into Drugs,

2nd Edition, Rodney

J.Y. Ho.



Pharmaceutical Biotechnology- Focus on Health & Medicine

Use of l iving things (e.g.bacteria) to create pharmaceutical products

Ronald A. Rader. Nature Biotechnology 26, 743 - 751 (2008)



Biotech Products• Larger M.W.

• Derived from living sources –human& animal tissues, cells &microorganisms

• Not easily characterized and refinedto high degree of purity.

• Often called by the same namedespite modifications in one or moreamino acid residues.

Insulin-human

Insulin-beef Insulin-pork

Insulin-aspart etc…

Key differences between biotech and chemical products

Traditional Drugs• M.W. typically < 1000Da

• Can be chemically synthesizedand purified to homogeneity

• Chemical modification usuallyleads to drastic changes inactivity and new drugs for new

uses.



Why Biopharmaceuticals?

Proteins are the basic building blocks of life that control diverse cellular andphysiological processes such as metabolism (energy expenditure), the immuneresponse (involved in fighting diseases) and memory and learning in the humanbrain. They play a critical role in the processes and interactions involved in

maintaining good health in the face of disease and ageing.

Therefore, protein-based drugs (biopharmaceuticals) may be a more natural

way of treating diseases compared to artificial small molecules.

Behave morepredictably

Fewer SE



• Majority (>95%) of biopharmaceuticals are protein products.

• Proteins are susceptible to protease degradation anddenaturation in biological fluids.

• Dosage forms that can be administered by IV, IM or SC routesare available.

• Distribution of proteins (macromolecules) to tissues is limited bythe permeability (porosity) of vasculatures

Absorption and Disposition?

Immunogenicity?

Challenges of using Biopharmaceuticals



1797: Jenner inoculates child with viral vaccine to protect him from smallpox

1857: Pasteur proposes that microbes cause fermentation.

1928: Penicillin is discovered by Flemmng.

1944: Avery demonstrates that DNA is the "transforming factor" and material of genes.

1953: Double helix structure of DNA is first described by Watson and Crick.

1973: Cohen and Boyer develop genetic engineering techniques to "cut and paste" DNA and reproduce the new DNA in

bacteria.1977: Genentech scientists and their collaborators produce the first human protein (somatostatin) in a bacterium (E. coli).

1978: Genentech scientists and their collaborators produce recombinant human insulin.

1979: Genentech scientists produce recombinant human growth hormone.

1981: First transgenic animal.1982: Eli Lilly and Company markets Genentech-licensed recombinant human insulin - the first such product on the market.

1983: Polymerase chain reaction (PCR) technique conceived (will become a major means of copying genes and genefragments).

1986: Genentech receives FDA approval for Protropin for growth hormone deficiency in children - the first biotech drug

manufactured and marketed by a biotech company.

1990: Human Genome Project (HGP), an international effort to map all the genes in the human body, is launched.

1994: BRCA1, the first breast cancer susceptibility gene, is discovered.

1995: The first full gene sequence of a living organism other than a virus, is completed for the bacterium Haemophilusinfluenzae.

2000: First draft of human genome sequence completed by the HGP and Celera Genomics.

Significant scientific milestones in biotechnology

http://www.accessexcellence.org/RC/AB/BC/



Capturing the History of Biotechnology<http://www.lifesciencesfoundation.org/index.html>



Recombinant DNA TechnologyGenetic Engineering

Gene Cloning

Media on “ Genetic Modification” :

http://www.pbs.org/wgbh/nova/genome/media/2809_q056_15.html



1. Isolation of gene of interest “ cut & paste”

2. Introduction of gene to expression vector

3. Transformation into host cells

4. Selection of the required sequence andpropagation of cells

5. Isolation & purification of protein

6. Formulation of protein product

Synthesis of a recombinant protein:

Six-Step ProcessGene cloning

21

Overview f Gene Cl nin



Overview of Gene Cloning

Ability to recombinesegments of DNA from

diverse sources into

new composite

molecules, orrecombinants.

2. Host cells

1. Vectors

3. Transformation &

transfection

4. Selection of

required rDNA

22



What is PCR?

• Polymerase Chain Reaction invented by Kary Mullis (1985)

• Produce millions of copies of a specific DNA sequence in a short time(approx 2h). This automated process bypasses the need to use bacteria for amplifying DNA.

• Use of a thermocycler.

23

Equipment that enables a mixture ofDNA and reagents to be incubated at aseries of temperatures that are varied ina preprogrammed manner.

Both gene cloning and PCR can provide pure samples of anindividual gene, separated from other genes.

“Cloning” DNA in a test-tubeCell-free DNA Cloning



PCR Methodology

Ingredients:DNA, Taq DNA polymerase, Specific primers,

nucleotides dNTPs (dATP, dCTP, dTTP, dGTP),

Mg2+

Steps:

1. Mixture is heated to 94 C (1 min).

2. Mixture is cooled down to 50

C-60

C to allowannealing of primers (1 min).

3. Temperature raised to 74 C to allow Taq DNA

polymerase to catalyze addition of nucleotides (1.5

min)4. The cycle of denaturation, annealing and synthesis

is repeated. After Cycle 30, > 1 billion identical

copies (230 = 1.07 x109).

denaturation

annealing

synthesis

24



• Principle of PCR in animation:http://www.youtube.com/watch?v=2KoLnIwoZKU

• CSI PCR in 60s

http://www.youtube.com/watch?v=6iFDphWXjw4

PCR Methodology



1. Sequences of primer annealing sites must be known.Easy to synthesize a primer with a predetermined sequence, but if the sequences of the annealing sites are unknown, appropriate primers cannot be made.

2. Length of DNA sequence that can be copied by PCR.5 kb can be copied fairly easily. Segments up to 40kb have to be dealt with by usingspecialized techniques.

3. Infidelity of DNA replicationTaq DNA polymerase has no 3′ → 5′ exonuclease (proofreading function). Error dueto base misincorporation during DNA replication.

High frequency: 1 kb sequence -> 20 cycles -> ~40% of the new DNA strands will

contain an incorrect nucleotide.Overcome by using alternative heat-stable DNA polymerases with 3′ → 5′ exonuclease activity. E.g. Pyrococcus furiosus (Pfu) DNA pol. Reduced error rate3.5%

26

Limitations of PCR



Other applications:

– Amplify DNA fragment for gene cloning

– Diagnostic applications:

1. PCR amplification of mutant alleles to determine if person is carrier of a genetic disease (sickle cell anaemia)

2. Early detection of disease to prevent onset, e.g. PCR amplification of DNA of a disease-causing virus.

– Forensic identification (DNA profiling) – Archaeology (sex identification)

– etc

– etc

– etc

27

Other Applications of PCR



Other Tools and Techniques for DNA work

• DNA purification from cells

• Gel electrophoresis

• Restriction enzymes• DNA sequencing

28

B i P i l ll DNA



Basic steps – Preparing total cell DNA

P ti f ll t t



Cell lysis

•Lysozyme – digests polymeric component of cell wal•EDTA (ethylenediamine tetraacetate) – removes Mg ions essential for preservingstructure of cell membrane and inhibits enzymes that could degrade DNA.

•SDS (sodium dodecyl sulfate) – Detergent; removes lipids and disrupts membranes.

Preparation of cell extract

DNA ifi ti f ll



DNA purification from cells

• Sources of total cell DNA to isolate and purify:Bacteria, plants or animals

• Common Methods:1. Contaminants removal by organic extraction:

Phenol or phenol/chloroform mixture precipitate proteins,leave nucleic acids in aqueous solutions.

31

DNA ifi ti f ll



2. Ion-exchange chromatography

Principle:

- DNA, RNA and some proteins(in decreasing order) arenegatively charged → bind topositively charged resin.

- Electrical attachment disruptedby salt → DNA can be removed

from resin and collected.

32

DNA purification from cells

C t ti d t f DNA l



Concentration of DNA by ethanol precipitation (in the presence of salt – monovalent cation e.g. Na+ and low temperature of < -20C):

• Ethanol mixed with a dilute DNA solution → DNA precipitates

and can be collected by centrifugation.

Measurement of DNA concentration:

• By UV spectrophotometry; absorbance measured at 260 nm. Abs (A260) of 1.0 = 50 g of double-stranded DNA per ml.

• Purity of DNA sample: ratio of absorbance at 260 and 280 nm(A260/A280) should be 1.8. If < 1.8, sample contaminatedwith protein or phenol.

Concentration and measurement of DNA samples

33

Gel Electrophoresis



Gel Electrophoresis

(a) Standard electrophoresis doesnot separate DNA fragments ofdifferent sizes, whereas (b) gelelectrophoresis does.

G l l t h i



Gel electrophoresis

• Principle: Electric current used to separate different-sized molecules in a

porous, sponge-like matrix. Smaller molecules move more

easily through the pores than larger-sized molecules.

• DNA characteristics:

Highly negatively charged due to phosphate groups in thebackbone; migrate towards the positive electrode.

DNA molecules of the same length will move through the gel at

the same rate.

35



Gel submerged in salt solution that

conducts electrici ty

1.

“ Tracking” solution addedto colorless DNA solution tovisually track DNAmigration through the gel

2.

Bands visualized by using

the ethidium bromide dyeor radioactivity(autoradiography).

3.

anode

36

Gel electrophoresis



Gel characteristics:• Agarose gel (made from

highly purified seaweed) or

polyacrylamide (PA) used toseparate DNA molecules fromseveral to 50,000 nucleotidesin length.

• Size resolution optimized bygel concentration

Separation characteristics for agarose andpolyacrylamide gels

Gel type Separation range (bp)

0.3% agarose

0.7% agarose

1.4% agarose

4% PA

10% PA

20% PA

50,000 to 1,000

20,000 to 300

6,000 to 300

1,000 to 100

500 to 25

50 to 1

Gel electrophoresis

37

Estimation of Sizes of DNA molecules



Estimation of Sizes of DNA molecules

38

(a) A rough estimate of fragment size can beobtained by eye.

(a) A more accurate measurement is gained byusing the mobility of the HindIII fragments toconstruct a calibration curve; the sizes ofunknown fragment can be determined fromthe distances they have migrated

Restriction Endonucleases



Restriction EndonucleasesGene cloning requires that DNA molecules be cut in a precise and

reproducible fashion.

39

Reasons for cutt ing DNA:• Single gene to be cloned may consist

only 2-3kb; to be cut out of the large

(>80kb) DNA molecules.• Large DNA to be broken down to produce

fragments small enough to be carried bythe vector.

Reasons for cutting vector:• Open up the circle so that new DNA can

be inserted.• To be cut at exactly same position on thecircle.




• Bacterial enzymes that recognize specific 4- to 8-bp sequences, calledrestriction sites, and then cleave both DNA strands at this site.

• Type I, II and III. Type II are the cutting enzymes impt in gene cloning.

• Three types of restriction fragments:

Blunt ends

Protruding (sticky) 3’ ends

Protruding (sticky) 5’ ends

• Sticky ends (also called cohesive ends) are complementary to and base pair with other fragments generated by the same restriction enzyme.

• Fragments can be covalently ligated by action of DNA ligase.






Note:Restriction enzymes with different recognition sites may produce same sticky ends.e.g. BamHI (GGATCC) and BglII (AGATCT) – GATC sticky ends

Sau3A (GATC)

Performing a restriction digest in the lab



Performing a restriction digest in the lab

DNA sequencing



DNA sequencing

• Objective: To determine sequences of bases in DNA.

• Steps involved:

1. Preparation of DNA fragmentsGenerate a set of overlapping fragments that terminate at

different bases and differ in length by 1 nucleotide. (Nested

fragments)

2. DNA sequencing

Maxam-Gilbert, Sanger-Coulson

3. Detection stepGel electrophoresis, Autoradiograph, Capillary electrophoresis

using fluorescent labels

43

Maxam-Gilbert (chemical) sequencing



Maxam Gilbert (chemical) sequencing

• Uses chemicals to cleave DNA at specific bases, resulting infragments of different lengths (chemical degradation of DNA).

• Advantages:

- Requires double-stranded DNA fragments,so need not be cloned in a plasmid vector.

- No primers needed.

- Direct sequencing of small fragments possible.

• Disadvantage:

Not suitable for large scale use (difficulty to be automated);

Chemicals used are toxic (pose health hazard).

44

Maxam-Gilbert (chemical) sequencing



G

G

1. Double-stranded fragment labelled at 5’ end with32P.

2. Labelled DNA sample denatured by heating(90°C). Results in breakdown of base

pairing and dissociation into two componentsstrands.

3. Strands were separated from one another bygel electrophoresis. One strand purified fromgel and divided into 4 samples, each of whichis treated with one of the cleavage reagents.

45

Maxam Gilbert (chemical) sequencing

Nucleotide Cleavage agent

G alone DMS, piperidine

A+G DMS, formic acid and piperidine

C+T Hydrazine, piperidine

C alone Hydrazine in high salt

G A+G C+T C



5. Bands not duplicated in A+G lane readas A. Bands not duplicated in C+Tread as T.

6. Sequence read from bottom of gelup (5’ to 3’).

46

4. Reactions are controlled so that eachlabelled strain is likely to be broken onceonly.

Parallel gel electrophoresisand autoradiography



Sanger-Coulson (chain termination) sequencing



g ( ) q g

• Requires single-stranded DNA

• Cloned into special vectors e.g. M13 vector or phagemids

• Double-stranded DNA converted to single stranded by

denaturation with alkali or boiling

• Thermal cycle sequencing using one primer

• Involves enzymatic DNA polymerase synthesis of a secondstrand of DNA, complementary to existing template.

• Chain terminates with the use of dideoxynucleotides.

48

Sanger-Coulson (chain termination) sequencing



g q g

49

Position where –OH of dNTP is replacedby –H;Phosphate group cannot be added to

elongate the chain

GC

C

3'

1. Incubation with DNA polymerase, primer, dATP,The parent sequence



C

A

A

ACC

G

5'

1. Incubation with DNA polymerase, primer, dATP,dCTP, dGTP, dTTP and dideoxyGTP

primer

3'

G

C

C

GC

A

A

ACC

G

5'

2. Short primerinitiatesreplicationprocess

3'

G

C

C

GC

G A

T A

T A

TC

GC

G

5'

3'

G

C

C

GC

G A

A

ACC

G

5'3. DNA polymerase catalyses

formation of complementarystrand

4. DNA biosynthesisstops when dideoxy

base added.

3'

G

C

C

GC

G A

T A

T A

TC

GC

G

G

5'

50

5. This process is repeated separately with the other 3 dideoxy bases (4 concurrent strand synthesis



CT AG

C

GG

T

T

TG

A

G

G

CC

A

A

AC

T

C

Sequenced DNA

Parent sequence

The autoradiogram providesthe sequence of thereplicated single-strandedDNA of the parentsequence

5’

3’5’

3’

p p p y y ( yreactions). Thus, 4 separate reactions result in 4 famil ies of terminated strands.

6. The double stranded DNA can be separated by heating, and the fragments are separated byelectrophoresis.

51

Automated DNA sequencing



Fluorescent probesare used for

automated sequencing(different fluorescentlabels attached toeach type of

dideoxynucleotide)

http://www.dnalc.org/ddnalc/resources/cycseq.html52

http://www.youtube.com/watch?v=SRWvn1mUNMA










y f m p

Six-Step Process Gene cloning

53

Vector for gene cloning (Cloning vector)



Basic features:

1. Able to replicate within host cell.2. Contains a site where DNA can be inserted (restriction sites).

3. Contains selective marker(s) e.g. antibiotic resistance

4. Relatively small, < 10 kb in size.

Examples:

Plasmids, bacteriophage, cosmids, bacterial and yeast artificialchromosomes (BAC/YAC), etc.

54

A DNA molecule that serves as a vehicle to transport a gene into hostcells.

5-10kb 12-20kb 35-45kb ~300kb ~1000kb

Plasmid vectors for use in E. coli



55

• Circular molecules of DNA that lead an independent existence in a host cell.• Found naturally in bacteria and some yeasts.

• Carry one or more genes responsible for useful characteristics displayed by

the host bacterium e.g. antibiotic resistance gene (selectable marker).• Generally dispensable (not essential for cell growth and division).

• Possess at least one DNA sequence that acts as origin of replication –

multiply independently of bacterial chromosome.• Or replicate by inserting themselves into the bacterial chromosome

(episome).

Replication strategies for plasmids



Non-integrative plasmid

Episome

56




pBR322 – “prototype” vector

used with E. coli.

Characteristics:

•Small size•2 antibiotic resistance genes

•Variety of restriction sites

•High copy no.

Naturally occurring plasmids extensively modif ied to produce vectors with

desired characteristics in genetic cloning.

57




58

pUC8 – a lac selection plasmid

Advantages over pBR322:•Higher copy no.

•Identification of recombinants a single step processPlating on agar containing ampicillin and X-gal

•Clustering of restriction sites

Selection of recombinant clones



Insertional inactivation of TetR:

•Transformed cells plated onto Ampagar, and replica plated onto Tet agar.

• Colonies that grow on Tet agar are AmpRTetR → non-recombinants.

• Colonies that carry inserted DNA do

not grow on Tet agar (AmpR

TetS

) → position on amp agar plate now known.

59

1. Insertional inactivation of an antibiotic resistance gene: AmpR gene has restrictions sites PstI, PvuI and ScaI. TetR gene has BamHI and SaII restriction sites.

Insertion of new DNA in one of these sites inactivates gene.

e.g. pBR322




2. Insertional inactivation of lacZ’ gene (Lac selection)• Unmodified lacZ gene codes for the –galactosidase ( –gal) enzyme (breaks

down lactose to glucose and galactose)

• Lac promoter (strong) is induced by the addition of IPTG [isopropyl-thiogalactoside]

Switches on gene transcription of lacZ gene to produce the –gal enzyme

• LacZ’ gene is modified from lacZ gene

Codes for part of the –gal enzyme (α-peptide portion); Used in mutantE.coli strains (E. coli lacZ’-) that have a modified lacZ gene that lacks thesegment of gene referred to as lacZ’ gene.

60

e.g. pUC8




In the presence of IPTG,

61




Using lacZ’ gene

Foreign gene is inserted in the

plasmid in a way that disrupts the

lacZ’ gene.

62




With the insertion of the foreign gene, the lacZ’ gene is disrupted and results in

unsuccessful production of the –gal enzyme component.

Lac selection

63

X-gal: lactose analog broken down byβ-gal to blue colour pdt

Bacteriophage vectors for use in E. coli



• Viruses that infect bacteria.• Consist DNA or RNA genome and a capsid (protein coat).

• Used to carry DNA fragments too large to be handled by

plasmids.

• M13 and phage commonly used as cloning vectors.

• Used to make DNA libraries.

64phage M13 phage




Phages classified as temperate or virulent, depending on their life cycles.

Lytic:

Enters bacteria,

produces morephages and killbacterial cells.

Lysogenic:

Integrate intochromosome,remains quiescentwithout killing cells.

• Virulent phages exhibit lytic life cycle only.• Temperate phages exhibit lysogenic life cycle, but may undergo lytic

response when conditions are suitable. – Eg: Lambda (

) phage65

Lysogenic infection cycle of bacteriophage



66

Episomal insertion

Triggering agents:UV, DNA damaging

agents




phage genome (49 kb) – impt features as a cloning vector

• Must be capable of lytic growth, other viral functions irrelevant.

• Genes involved in lysogenic pathway and other viral genes not essential for

lytic pathway removed → Deleted genome non-lysogenic, can follow onlylytic infection cycle. Desirable feature for cloning vector (induction notneeded before plaques are formed).

• Replaced with DNA (12 – 20 kb) to be cloned.

Foreign gene can be

inserted

67

2 f i f DNA l l li d i l f




68

•2 conformations of DNA molecule: linear and circular forms.

• Linear form: consists of two complementary strands of DNA withshort single-stranded 12-nucleotide stretch at two free ends.

• “Sticky” or “cohesive” ends called cos sites




69

cos sites

1.Allows linear DNA moleculeinjected into host cell to circularize.

2.Rolling circle mechanism of replication results in a catenaneconsisting of a series of linear

genomes joined together at the cossites.

Cos sites recognized by

endonucleases to cleave thecatenane to produce individual

genomes.

http://www.youtube.com/watch?v=ehbZpo8oXSs




• Use it like a plasmid vector cloning, but not efficient.

• Modifications required for greater number of recombinants.

70




Recombinant DNA

In vitro phage assembly

+ protein forpackagingDNA)

• Use as recombinant phage

71

E. coli cells infectedwith λ mutant – aprotein required forpackaging λ DNAinto preassembledphage headsmissing.

Cells accumulate“empty” heads.

Other Cloning Vectors



• Cosmid• Hybrid between plasmid and λ vector.

• Size of insert: 40-45 kb.

• Bacterial Artificial Chromosome (BAC)

• Based on the F. plasmid from E. coli

which is much larger than the standardplasmid vectors.

• Size of insert: 300 kb.

• Yeast Artif icial Vector (YAC)

• Vector containing yeast genes.

• Size of insert: 1 Mb.

72










Six-Step Process Gene cloning

73

Host Cells

I t &B t i Y t T i Pl t



• Insufficient foldingof complexproteins of higherorganisms –inclusion bodies

• Lack of post-translationalmodifications

• Endotoxins

Insect &Mammalian

Bacteria Yeast Transgenic Plants& Animals

• Post-translationalmodificationsdiffers frommammalian cells

• Problematic cell

disruption• Protease thatdegrade foreignproteins

• Laboriousconstruction ofover-expressingstrains

• Expensive media

• Low growth rates

• Difficult scale-up

• Longdevelopmentalcycles

• Contaminationproblems

– Animal viruses

– prions

74e.g. E.coli

CHO cells “Pharming”

Production of Recombinant Protein in E.coli



Source of gene for cloning

Source material: Nucleic acid molecules in the form of mRNA or genomic DNA



Source material: Nucleic acid molecules in the form of mRNA or genomic DNA

mRNA Genomic DNA

Represents the coding sequence of a gene,

with any introns removed during RNAprocessing → Production of a recombinantprotein > straightforward.

Represents the genetic information that isbeing expressed by the particular cell typefrom which it is prepared.

If gene of interest is highly expressed, mRNAwill be in abundance, making isolation ofclones easier.

Contains non-coding DNA such as

introns, control regions and repetitivesequences.

Represents the full complement of DNAcontained in the genome of a cell ororganism.

For studies on control of geneexpression, isolation of controlsequences is necessary, genomic DNAis the only alternative.

76

Cloning from mRNA: cDNA synthesisIt is not possible to clone mRNA directly, so i t has to be converted into cDNA



(complementary DNA) before being inserted into a suitable vector.

mRNA

Oligo(dT) primer binds to

poly(A) tract at 3’end

RTase synthesizes a copyof mRNA to produce acDNA-mRNA hybrid mRNA breakdown with alkali or

RNaseH

Duplication by DNA polymeraseWhat primers to use?

Double strandedcDNA

“ cDNA” http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

RNA-primingusing oligo-dT

ss cDNA(1st strand cDNA)

77

How to obtainmRNA from cells?

l1 3 introduction dna 2014

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