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 base-paired 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

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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.

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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/sn-PCR%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 32

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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 post-amplification 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

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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.

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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