Recombinant DNA Technology

Definition of recombinant DNA

Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

DNA fragments are usually derived from different biological sources

ApplicationsGene isolation/purification/synthesisSequencing/Genomics/ProteomicsPolymerase chain reaction (PCR)Mutagenesis (reverse genetics)Expression analyses (transcriptional and

translational levels)Restriction fragment length polymorphisms

(RFLPs)Biochemistry/ Molecular modelingHigh throughput screeningCombinatorial chemistryGene therapy

Recombinant VaccinesGenetically modified cropsBiosensorsMonoclonal antibodiesCell/tissue cultureXenotransplantationBioremediationProduction of next generation antibioticsForensicsBioterrorism detection

Common steps involved in isolating a particular DNA fragment from a complex mixture 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

3. Introduction of recombinant DNA molecule into an appropriate host cellTransformation or transfectionEach cell receiving rDNA = CLONEMay have thousands of copies of rDNA

molecules/cell after DNA replicationAs host cell divides, rDNA partitioned into

daughter cells

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

molecular analysesInvestigate organization of genesStructure/functionActivationProcessing

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

II. Restriction Endonucleases

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.

Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses.

Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity.

Collectively, these two processes form the restriction modification system.

To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA.

While recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous base sequences that read the same backwards and forwards.

In theory, there are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on the same DNA strand (i.e., single stranded) as in GTAATG.

The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., double stranded) as in GTATAC (Notice that GTATAC is complementary to CATATG).

The inverted repeat is more common and has greater biological importance than the mirror-like.

Palindromes in DNA sequences

Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands.

5’

5’

3’

3’

Restriction endonucleases are categorized into three or four general groups based on their composition and enzyme cofactor requirements.

They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements:

Type I enzymes cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase activities.

Type II enzymes cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase.

Type III enzymes cleave at sites a short distance from recognition site; require ATP (but doesn't hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exist as part of a complex with a modification methylase .

Type IV enzymes target methylated DNA.

Sticky end

Sticky end

III. Vectors for Gene Cloning

A. Requirements of a vector to serve as 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.

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.

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

B. Main types of vectors

Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), retrovirus, baculovirus vector……

Depends on nature of protocol or experiment

Type of host cell to accommodate rDNAProkaryoticEukaryotic

C. Choice of vector

Plasmid vectors

Advantages:Small, easy to handleStraightforward selection strategiesUseful for cloning small DNA fragments (< 10kbp)

Disadvantages:Less useful for cloning large DNA fragments (> 10kbp)

Plasmid vectors are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules.

1. Contains an origin of replication, allowing for replication independent of host’s genome.

2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistanceblue-white screening

3. Contains a multiple cloning site (MCS)4. Easy to be isolated from the host cell.

A plasmid vector for cloning A plasmid vector for cloning

Plasmid Plasmid vectorsvectors

ExamplespBR322

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

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

ColE1 ORI

pUC18Derivative of pBR322Advantages 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”

E. Lambda vector

Bacteriophage lambda infects E. coliDouble-stranded, linear DNA vector – suitable

for library constructionCan accommodate large segments of foreign

DNACentral 1/3 = “stuffer” fragment

Can be substituted with any DNA fragment of similar size without affecting ability of lambda to package itself and infect E. coli

Accommodates ~15kbp of foreign DNAForeign DNA is ligated to Left and Right Arms of

lambda Then either: 1) Transfected into E. coli as naked DNA, or2) Packaged in vitro by combining with phage protein

components (heads and tails) (more efficient, but labor intensive and expensive)

Left arm:head & tail proteins

Right arm:DNA synthesisregulationhost lysis

Deleted central region:integration &

excisionregulation

F. Cosmid vectors

Hybrid molecules containing components of both lambda and plasmid DNALambda components: COS sequences

(required for in vitro packaging into phage coats)

Plasmid DNA components: ORI + Antibiotic resistance gene

Cloning sites will be part of vectorrDNA is packaged using extracts of coat

and tail proteins derived from normal lambda components BUT cannot be packaged after introduced into host cell because rDNA does not encode the genes required for coat proteins

After infection of E. coli, rDNA molecules replicate as plasmids

Very large inserts can be accommodated by cosmids (up to 35-45 kbp)

Disadvantages:Not easy to handle very large plasmids (~ 50 kbp

ZAP

G. 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 typesExamples:

E. coli yeast cellsE. coli human cell lines

Selectable markers and cloning sites

H. Bacterial artificial 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 presentSelectable markers and cloning sites

availableOther useful features:

SP6 and T7 promoters Direct RNA synthesis RNA probes for hybridization experimentsRNA for in vitro translation

BAC vector

oriS and oriE mediate replication

parA and parB maintain single copy number

ChloramphenicolR marker

I. Yeast artificial chromosomes (YACs)

Hybrid molecule containing components of yeast, protozoa and bacterial plasmidsYeast:

ORI = ARS (autonomously replicating sequence)Selectable markers on each arm (TRP1 and

URA3)Yeast centromere

Protozoa= TetrahymenaTelomere sequences (yeast telomeres may also

be used)Bacterial plasmid

PolylinkerCan accommodate >1Mb (1000kbp =

106 bp)

YAC vector

Capable of carrying inserts of 200 - 2000 kbp in yeast

telomere telomerecentromere

URA3ARS HIS3

replicationorigin

markers

largeinserts

What determines the choice vector?

insert size

vector sizevector size

restriction sitesrestriction sites

copy numbercopy number

cloning efficiencycloning efficiency

ability to screen for insertsability to screen for inserts

Expression vector

J. Human artificial chromosomes

Developed in 1997 – synthetic, self-replicating

~1/10 size of normal chromosomeMicrochromosome that passes to cells

during mitosisContains:

ORICentromereTelomereProtective cap of repeating DNA sequences

at ends of chromosome (protects from shortening during mitosis)

Histones provided by host cell

IV. Constructing Genomic and cDNA Libraries

A. Definition

A cloned set of rDNA fragments representing either the entire genome of an organism (Genomic library) or the genes transcribed in a particular eukaryotic cell type (cDNA library)

rDNA fragments generated using restriction endonucleases

rDNA fragments ligated to appropriate cloning vector

B. Genomic libraries

Commonly bacteriophage lambda used as the vector“Stuffer fragment” removed and replaced with 15-

17kbp fragments of libraryCosmids and YACs may also be used as

vectorsContains at least one copy of all DNA

fragments in the complete libraryScreened using nucleic acid probes to

identify specific genesSubcloning is usually necessary for detailed

analysis of genesPreparation of genomic library in

bacteriophage lambda vector

Determination of library size:The larger the fragments that are cloned in a

particular vector the smaller the overall size of the library

N = ln (1-P)/ ln (1-f)

N = Number of required clonesP = probability of recovering a desired DNA

sequence (P= 0.99)f = fraction of the genome present in each

clone (insert)

Example: Human genome = 3.2 x 106 kbp = 3.2 x 10 9 bp

Lambda vector can accommodate 17kbp inserts

N = ln (1 – 0.99) ln [1 – (1.7 x 104 bp insert) 3.2 x 109 bp genome]

N = 8.22 x 105 plaques required in library

Usually researchers will make genomic libraries 2 – 2.5x the size required using this equation.

Human Genome Project (HGP)Entire human genome has been sequenced

(April 2000)Project began in 1990 – Joint Venture

Human Genome Organization (HuGO) (USA, UK, France, Japan mainly)

CELERAThis topic will explored in more detail later in

the course.

C. cDNA librariesmRNA represents genes that are actively

transcribed (or expressed) at any given time in a particular cell typeSmall subsets of sequences found in a

genomic libraryEukaryotic mRNA = polyadenylated and

introns have been removed This is the starting point!

mRNA converted into a DNA copy (=cDNA) using a series of enzymatic reactions and oligonucleotidesPrimer, reverse transcriptase, DNA polymerase I,

S1 nuclease, linkers, restriction enzymes, vectorSize of library depends on abundance of

messageBacteriophage lambda insertion vectors or

plasmids are used for cloningThe choice depends upon:

Abundance of mRNASize of desired libraryScreening method

Method – cDNA Synthesis and Cloning into a Plasmid Vector1. mRNA must be separated from other

cellular constituents before 1st strand cDNA synthesis is carried outRNA is first purified and DNA is eliminated Isolation of poly(A) RNA using Oligo (dT)

cellulosePoly (A) tails of mRNA hybridize to oligo (dT)

cellulose resin via column chromatographyrRNA and tRNA do not bind and are eluted

After extensive washing of the column, then mRNA is eluted by dropping salt concentration, precipitated, washed and quantitated

2. mRNA is combined with an oligo (dT)15-18 synthetic primer which binds to the 3’ end of mRNA

3. Reverse transcriptase is added and synthesis of a DNA copy of the mRNA takes place beginning at 3’ –OH of oligo (dT) primer, extending the cDNA in the 5’ to 3’ direction

4. Alkali treatment degades the mRNA template leaving the first strand of cDNA

5. A hairpin loop forms on the first strand cDNA product.

6. DNA polymerase I is added which extends the hairpin loop back in the 5’ to 3’ direction to complete the second strand cDNA product

7. S1 nuclease digests single stranded ends and the hairpin loop leaving a ds cDNA product with flush ends.

8. Lambda exonuclease is added to nibble back a few nucleotides from the ends to generate short single-stranded overhangs.

9. Terminal deoxynucleotidyl transferase (TdT) is added plus deoxythymidine triphosphate generating strings of Ts at ends of molecules.Alternatively synthetic DNA linkers can be ligated

at this stage.

10. cDNA can be cloned into a plasmid with complementary strings of A’s by hydrogen bonding and DNA ligase. If alternative is used above, then the plasmid is

digested with appropriate restriction enzyme to produce compatible sticky ends.

11. Recombinant plasmids are transformed into E. coli to produce cDNA library.

12. Screening cDNA libraries is carried out using nucleic acid probes, degenerate oligonucleotide probes, or antibodies.Dependent on resources available and vector used.