Download Part II: Recombinant DNA Technology

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
Applications
 Gene isolation/purification/synthesis
 Sequencing/Genomics/Proteomics
 Polymerase chain reaction (PCR)
 Mutagenesis (reverse genetics)
 Expression analyses (transcriptional and translational levels)
 Restriction fragment length polymorphisms (RFLPs)
 Biochemistry/ Molecular modeling
 High throughput screening
 Combinatorial chemistry
 Gene therapy
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Recombinant Vaccines
Genetically modified crops
Biosensors
Monoclonal antibodies
Cell/tissue culture
Xenotransplantation
Bioremediation
Production of next generation antibiotics
Forensics
Bioterrorism 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 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
 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
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Investigate organization of genes
Structure/function
Activation
Processing
 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
5
’
3’
3’
5
’
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.
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 Sadenosyl-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……
C. Choice of vector
 Depends on nature of protocol or experiment
 Type of host cell to accommodate rDNA
 Prokaryotic
 Eukaryotic
Plasmid vectors
Plasmid vectors are double-stranded, circular, selfreplicating, extra-chromosomal DNA molecules.
 Advantages:
 Small, easy to handle
 Straightforward selection strategies
 Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
 Less useful for cloning large DNA fragments
(> 10kbp)
A plasmid vector for cloning
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 resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Plasmid vectors
 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)
 ColE1 ORI
 pUC18
 Derivative of pBR322
 Advantages over pBR322:
 Smaller – so can accommodate larger DNA fragments during cloning (510kbp)
 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 (l) infects E. coli
 Double-stranded, linear DNA vector – suitable for library
construction
 Can accommodate large segments of foreign DNA
 Central 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 DNA
 Foreign DNA is ligated to Left and Right Arms of lambdaThen either:
 1) Transfected into E. coli as naked DNA, or
 2) 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 synthesis
 regulation
 host lysis
 Deleted central
region:
 integration & excision
 regulation
F. Cosmid vectors
 Hybrid molecules containing components of both
lambda and plasmid DNA
 Lambda components: COS sequences (required for in vitro
packaging into phage coats)
 Plasmid DNA components: ORI + Antibiotic resistance gene
 Cloning sites will be part of vector
 rDNA 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
l 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
types
 Examples:
 E. coli  yeast cells
 E. 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 present
 Selectable markers and cloning sites available
 Other useful features:
 SP6 and T7 promoters
 Direct RNA synthesis
 RNA probes for hybridization experiments
 RNA 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 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)
YAC vector
large
inserts
URA3
HIS3
ARS
telomere
telomere
centromere markers
replication
origin
 Capable of carrying inserts of 200 - 2000 kbp
in yeast
What determines the choice vector?
 insert size
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vector size
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restriction sites
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copy number
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cloning efficiency
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ability to screen for inserts
Expression vector
J. Human artificial chromosomes
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Developed in 1997 – synthetic, self-replicating
~1/10 size of normal chromosome
Microchromosome 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
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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 library
 Cosmids and YACs may also be used as vectors
 Contains at least one copy of all DNA fragments in the
complete library
 Screened using nucleic acid probes to identify specific
genes
 Subcloning is usually necessary for detailed analysis of
genes
 Preparation 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 clones
 P = 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)
 CELERA
 This topic will explored in more detail later in the course.
C. cDNA libraries
 mRNA represents genes that are actively transcribed (or
expressed) at any given time in a particular cell type
 Small subsets of sequences found in a genomic library
 Eukaryotic 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 oligonucleotides
 Primer, reverse transcriptase, DNA polymerase I, S1 nuclease,
linkers, restriction enzymes, vector
 Size of library depends on abundance of message
 Bacteriophage lambda insertion vectors or plasmids are used
for cloning
 The choice depends upon:
 Abundance of mRNA
 Size of desired library
 Screening method
Method – cDNA Synthesis and Cloning into
a Plasmid Vector
 1. mRNA must be separated from other cellular
constituents before 1st strand cDNA synthesis is carried
out
 RNA is first purified and DNA is eliminated
 Isolation of poly(A) RNA using Oligo (dT) cellulose
 Poly (A) tails of mRNA hybridize to oligo (dT) cellulose resin
via column chromatography
 rRNA 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.