Transcript Chapter 4 part I

Chemical Synthesis,
Amplification, and Sequencing
of DNA (Part I)
Chemical synthesis of DNA
• DNA synthesis, amplification and sequencing
derived from knowledge of DNA structure
and replication.
• These methods are essential for isolating ,
characterizing, and expressing cloned gene.
• New protocols spawn novels experiments,
and laboratory procedures that were at one
time difficult to implement become much
easier to perform.
Flowchart for the
synthesis of DNA
oligonucleotides
• After n cycles, a
single-stranded
piece of DNA with
n+1 nucleotodes is
produced
Starting complex for the
chemical synthesis of DNA
strand
• The initial nucleoside has
a protective DMT group
attached to the 5’.
• A spacer molecule
attached to the 3’ hydroxyl
group of the deoxyribose.
• The spacer unit is
attached to a solid support,
CPG bead.
Phosphoramidites for each of the 4 bases
• A diisopropylamine group is attached to the 3’ phosphite
group of the nucleoside.
• A β-cyanoethyl groups protects the 3’ phosphite group of the
deoxyribose.
• DMT group attached to the 5’ hydroxyl group of the sugar.
Phosphoramidites for each of the 4 bases
• Before their introduction into the reaction column, the amino
groups of the adenine, guanine, and cytosine are derivatized
by the addition of benzoyl, isobutyryl, and benzoyl groups.
• Thymine is not treated because it lacks an amino group.
• This process prevents undesirable side reactions during chain
growth.
Detritylation
• The 5’ DMT group is removed from the attach nucleoside by
treatment of trichloroacetic acid (TCA) to yield reactive 5’
hydroxyl group.
• Then, the column is washed with acetonitrile to remove TCA
and then with argon to remove acetonitrile.
Activation and coupling
• After TCA the next prescribed base
and tetrazole are introduced
simultaneously.
• The tetrazole activates the
phosphoramidite so that its 3’
phosphite forms a covalent bond with
5’ hydroxyl group of the initial
nucleoside.
Capping
• The available 5’ hydroxyl group of unreacted
detritylated nucleosides are acetylated to prevent
them from participating in the coupling reaction of the
next cycle.
Oxidation
• The phosphite triester is oxidized with an iodine
mixture to form more stable pentavalent phosphate
triester.
Chemical synthesis of DNA
• After the oxidation and subsequent wash of the
reaction column, the cycle is repeated with each
successive phosphoramidite until the last programmed
residue has been added to the growing chain.
• The newly synthesized DNA strands are bound to the
CPG beads.
• Each phosphate triester contains a β-cyanoethyl
group.
• Every G, C, and A carries its amino-protecting group.
• The 5’ terminus of the last nucleotide has a DMT
group.
Chemical synthesis of DNA
• The β-cyanoethyl groups are removed by a chemical
treatment in the reaction column.
• The DNA strands are then cleaved from spacer
molecule, leaving 3’ hydroxyl terminus.
• The DNA is eluted from the reaction column.
• The benzoyl and isobutyryl groups are stripped from
the bases and the DNA is detritylated to remove DMT.
• The 5’ terminus of the DNA strand is phosphorelated
either by T4 polynucleotide kinase reaction or by a
chemical procedure.
• The phosphorelation can also be carried out while still
bound to the support.
Overall yields of oligonucleotides
• The coupling efficiency determined by spectrophotometry
should be greater than 98%.
• It may be necessary to purify the final product by
reverse-phase HPLC or gel electrophoresis to separate
longer target from the shorter “failure” sequences.
Uses of synthesized oligonucleotides
• To use as a single-stranded hybridization
oligonucleotide probes.
• The sequence can be formulated by deducing the
codons from the aminoacid sequence of a protein.
• These probes can be used to screen a genomic library
for the gene.
• Due to the codon redundancy, especially at the third
position, a single probe may not hybridize with the
heterologous sequence.
• A set of mixed (degenerated) probes is often used to
screen a genomic library.
• All possible DNA sequences deduced from a
protein sequence are highly complementary to
a heterologous gene.
• Typical linker and adapter
sequence; 6-mer and 8-mer
EcoRI linkers; BamHI-SmaI
adapter
• Linkers are added to
source DNA to facilitate
cloning into vector (Fig
4.10)
• Adaptors are
variants of linkers
that are often used to
create novel cloning
sites in vector (Fig.
4.11)
Uses of synthesized oligonucleotides
• Oligonucleotides are the key components for
assembling genes.
• The applications are including large-scale production
of proteins, testing protein function after changing
specific codons, and creating nucleotide sequences
that encode proteins with novel properites.
• The production of short genes (60-80 bp) can be
accomplished by synthesizing the complementary
strands and then annealing them.
• However, special strategies must be devised for larger
gene.
• Individual nucleotides
are synthesized.
• Their sequences are
designed to enable them
to form a stable
molecule, with basepaired regions separated
by gaps.
• The gaps are filled and
the nicks are sealed with
T4 DNA ligase.
Polymerase Chain Reaction (PCR)
• Two synthetic oligonucleotide primers (~20 mers
each) that are complementary to regions on opposite
strands that flank the target DNA sequence which
3’OH pointed toward each other after annealing.
• A template sequence in a DNA sample that lies
between the primer binding sites (100-35,000 bp.)
• A thermostable DNA polymerase that can withstand
being heated to 95º C or higher and copies the DNA
template with high fidelity.
• The 4 deoxyribonucleotides (dNTPs).
Polymerase Chain Reaction
PCR is a simple method for making multiple copies
of a DNA sequence, such as a gene
 Denaturing: double-stranded fragments of DNA heated
to denature (break H-bonds) them into single strands
 Annealing: short primers (15-20 bases) matching 3’
end of DNA fragments and sufficient quantities of the
four dNTPs are added
 Extending: DNA polymerase catalyzes synthesis of
new DNA strands
Second PCR cycle
• The templates for this
cycle are long templates
synthesized during the
first PCR cycle and the
original DNA strands.
Third PCR cycle
• During the renaturation
step, the primer
sequences hybridize to
complementary regions
of original, long-template,
and short-template
strands.
Thirtieth PCR cycle
• By the 30th cycle, the
population of DNA
molecules in a reaction
tube consists almost
entirely of short (target)
strands.
Polymerase Chain Reaction
 Repeating the cycle many times leads
to a huge increase (amplification) in the
number of copies of DNA
 1 million copies of a gene can be made
in just 20 PCR cycles
Thermocycler
http://www.river-joy.com/DNA%20Lab%20Tour/snPCR%20Machine.jpg
Polymerase Chain Reaction
 PCR has had an enormous
impact on genetic research
 The inventor, biochemist
Kary Mullis, earned a
1993 Nobel prize in
Chemistry.
 PCR requires a DNA
polymerase that survives high
heat extracted from hot
springs bacterium, Thermus
aquaticus (Taq polymerase)
• PCR can amplify sequences from a single
DNA molecule
PCR Considerations
 DNA Template
 Primer Design
 DNA Polymerase
 Thermo Cycling Program
 Reaction Conditions
 Controls
PCR Considerations
 DNA Template
- PCR is a very robust technique
- DNA preparation is relatively simple
- Target DNA sequences do not have to be
isolated from other DNA
- Great care must be taken to avoid contamination
of samples in case of forensics study
- The starting DNA template may be isolated from
prokaryotic or eukaryotic organisms, or such
exotic organisms as viruses
PCR Considerations
 Primer Design
- DNA primers of 18-24 nucleotides are commonly
used for PCR
- They may also be degenerate at one or more
nucleotides to permit amplification of target DNAs
of more variable sequence
- Degenerate primers are mixtures of primers with
different nucleotides occurring at the same primer
position on different primer molecules
PCR Considerations
 Primer Design
- GC content in the range of 40-60%
- Relatively high melting temperature (Tm; ideally
60-70°C)
- Use primer pairs with Tm not more than 5°C
different
- Tm = 4(G+C) + 2(A+T)°C.
- The annealing temperature for use in PCR can
further be approximated by subtracting 5°C from
the Tm
PCR Considerations
 Primer Design
- Long stretches of a single nucleotide
(as 5’-AAAAAAA-3’) should be avoided
- Primers should not be self-complementary nor
complementary to the other primer used in the
PCR reaction
- Restriction enzyme cleavage sites may be added
to the 5’ ends of primers to facilitate postamplification cloning of PCR products
PCR Considerations
 DNA Polymerase
- PCR was developed using Taq DNA polymerase
- Taq polymerase has no proofreading ability
- It is known to introduce an incorrect nucleotide
for about every 2x104 nucleotides
- DNA polymerases (such as Pfu) that incorporate
a proofreading ability may be used in place of Taq
PCR Considerations
 Thermo Cycling Program
- Denaturation; generally 94°C for 5 minutes
- Followed by 25-30 repetitive cycles of
denaturating, annealing and extending
- Denaturing; 94°C for 30 seconds
- Annealing; 30-65°C for 30 seconds
- Extending; 65-75°C for 2-5 minutes
PCR Considerations
 Reaction Conditions
- Nucleotide
- Magnesium
- Salt Concentrations
- Number of cycles and temperature profiles
PCR Considerations
 Controls
- Many opportunities for contamination
- Aerosol resistant pipette tips
- Omit DNA template and/or one or both primers
- Control reactions with known positive or negative
samples
PCR amplification of full-length cDNAs
• First strand cDNA is synthesized by reverse
transcriptase using oligo(dT).
• The terminal transferase activity of RT adds dCs to
the end of first strand cDNA.
• Primer-dG oligomer acts as a template for RT to
extend first strand cDNA.
• Forward and reverse primers that have the same
sequences as the primer-dG and oligo(dT) primer are
added.
• Full-length double stranded cDNA are generated by
PCR amplification.
PCR amplification of full-length cDNAs
Gene synthesis by PCR
• Overlapping oligonucleotides (A and B) are filled in
during DNA synthesis.
• Oligonucleotides (C and D) that are complementary to
the ends of the product of the first PCR cycle are added.
• Overlapping molecules are formed after denaturation and
renaturation, and the recessed ends are filled.
• Oligonucleotides (E and F) that overlapped the ends of
the second PCR cycle product are added and the third
PCR cycle is initiated.
• The final PCR product is a doubled stranded DNA
molecule with a specified sequence of nucleotides.
Variation on Standard PCR
 RT PCR
 Real Time PCR
RT - PCR
 Using RNA Template
 Enzyme reverse transcriptase is used to make a
cDNA copy of the starting RNA
 The choice of primer depends on the starting RNA
template and characteristics/knowledge of the target
for amplification
 RT-PCR follows the same steps found in standard
PCR from a DNA template
Real Time PCR
 Powerful technique for rapid and accurate
quantification of DNA or RNA
 Use a labeled oligonucleotide probe, often
referred to as a molecular beacon
 A short (30 to 40-bp) oligonucleotide synthesized
to be complementary to an internal sequence of
the target DNA amplified
 It carries a fluorescent reporter dye at its 5’ end
and a quenching dye at its 3’ end
Real Time PCR
 The 5’ and 3’ ends are
complementary so that
the beacon folds onto
itself forming a hairpin
structure
 The folding of the
beacon prevents any
emission of energy as
a fluorescent signal
when irradiated
Real Time PCR
 During the denaturation step in the PCR cycle, the
hairpin melts forming a linear molecule which will
hybridize to the target strand during the annealing
step
 The reporter and quencher dyes are now separated
allowing the reporter to fluoresce when irradiated
Real Time PCR
 The fluorescent signal is proportional to the quantity
of target DNA in the PCR reaction since the reporter
fluoresces only when hybridized to the target
sequence.
 The beacon is displaced from the target DNA and
available for the next cycle as the DNA polymerase
moves along the template strand.
Real Time PCR
 Real-time PCR with a probe designed for specific
target DNA can be used for detection and
quantification of plant and animal diseases.
 Multiplex PCR using multiple beacons, each with
different fluorescent labels, can be used to report on
PCR products amplified from different targets in a
single reaction