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Introduction

In 1987, Powell et al. described a technique that revolutionized gene cloning. The early 21st century witnessed an increase in the generation of synthetic genes to address limitations in classical cloning methods, marking a profound shift to a more integrative way of engineering molecular genetic research.

Classical cloning relies on the use of one or more enzymatic reactions. Optimization of the different reaction conditions must be carried out to allow the copying of variable amounts of template. Advances in enzyme technology enhanced the ability of different enzymes to deliver sequence-specific and organism-specific genes. Increasingly, buffer and salt conditions were optimized permitting a variety of non-conventional approaches. Gene synthesis was designed to circumvent the limitations of gene cloning. It produces genes of sufficient quality with fragment lengths of 10 kb or longer. Gene synthesis is nearly error-free because sequences are verified and mutations produced during the synthesis process are corrected. Synthesized genes can also be checked for their ability to express proteins, and gene function can be analyzed at both the transcriptional and translational level. Gene synthesis is useful in the creation of specialized cDNA libraries, large-scale production of microarray-ready cDNA, the design of gene therapy vectors, and the synthesis of gene variants.

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When to choose classical cloning methods?

Gene synthesis requires that the sequence of the gene be already known using conventional cloning methods. When sequence errors are found in databases, these errors will also be included in the synthesized gene. For example, the gene sequence of native AMV in the databases contained three errors, which could be verified using classical cloning methods.

In addition, gene synthesis procedures strongly depend on the quality of the oligonucleotides used. Gene synthesis limitations may, thus make classical cloning the preferred option. Oligonucleotides synthesized for gene synthesis must be linked to each other using specific annealing, standard ligation, or polymerase reactions; in addition, subcloning may be required. Classical cloning methods have the potential of discovering spatiotemporally expressed genes and naturally occurring SNPs, when the appropriate enzymes are used. Enzymes with proofreading capabilities and a sustained functional half life can produce cloned genes that are a correct and exact replica of an expressed transcript and a full-length gene.

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What is important regarding nucleic acid purification for cloning?

• Obtain high purity RNA that is the starting point for successful protein expression.

Tip: Obtain high sensitivity, reproducibility, and specificity for downstream cloning applications using the Roche High Pure RNA Isolation Kit or High Pure RNA Tissue Kit. These kits integrate on-column DNase treatment for eliminating genomic DNA, avoiding nonspecific side products that can interfere with subsequent cloning steps. Resources are conserved using a single kit for multiple applications and sample types.

• Proper handling and extraction of the starting material is extremely important, specifically when handling RNA samples. At all times, avoid high temperatures (which can result in denaturation) and degradation caused by RNases found in the sample.

Tip: Roche's Protector RNase Inhibitor protects RNA from degradation in reverse transcription reactions up to +60°C. It is optimally combined with Transcriptor High Fidelity cDNA Synthesis Kit even when transcribing GC-rich RNA at elevated temperatures.

• Always ensure that the starting material (cDNA, mRNA) is of the optimal quality, thus maintaining the best possible PCR performance. Starting material integrity and amount should be verified using gel electrophoresis.

• After cDNA synthesis, amplification, or enzymatic modification, DNA fragments may be purified for subsequent cloning. These DNA fragments can be repurified using agarose gel electrophoresis or simply used with the reaction mix of the previous enzymatic reaction.

Tip: Use the High Pure PCR Product Purification Kit or the High Pure PCR Cleanup Micro Kit for the purification of DNA fragments for efficient and specific protein expression. Eliminate primer-dimers less than 100 bp in size, and concentrate the nucleic acid in 10 µl using the High Pure PCR Cleanup Micro Kit. Purify DNA fragments out of agarose slices using the High Pure PCR Product Purification Kit.

• Reduce cloning steps and perform subsequent restriction enzyme digests directly in the Pwo SuperYield DNA Polymerase, dNTPack as described in the Related Documents.

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How to minimize errors and frame shifts?

High fidelity, proofreading PCR enzymes have been available for many years. Compared to other DNA polymerases used in nucleic acid analysis, retroviral reverse transcriptases commonly used for cDNA synthesis exhibit a higher error rate. This decreased accuracy leads to a significant number of base exchanges and frame shifts, which are propagated in subsequent PCR reactions.

• Make use of proofreading enzymes for reverse transcription and PCR reactions that ensure the exact replica of the gene expressed is copied, thus preventing nucleotide misincorporation.

Tip: Roche's optimized Transcriptor High Fidelity cDNA Synthesis Kit is a proofreading reverse transcriptase with seven-fold higher accuracy than normal reverse transcriptases to overcome nucleotide misincorporation. Achieve low error rates and prevent frame shifts in combination with a proofreading DNA polymerase such as Pwo SuperYield DNA Polymerase with 18-fold higher accuracy.

• Carefully designed primers ensure complementary sequence binding without frame shifts and incorrectly inserted nucleotides.

• Always use balanced amounts of all four dNTPs to minimize polymerase error rates; unequal concentrations can reduce the fidelity of thermostable DNA polymerases.

Tip: Choose the >99% pure and balanced PCR-grade nucleotides included in a separate vial in the Pwo SuperYield DNA Polymerase, dNTPack.

• Ensure optimal PCR amplification conditions.

• Ensure optimal growth conditions for cells and avoid inducing apoptosis (which results in DNA breaks).

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What is important for obtaining full-length clones?

• During cDNA synthesis, avoid mispriming of the oligo(dT) primer in the long poly (A) tail.

Tip: Use the anchored-oligo(dT)18 primer of the Transcriptor High Fidelity cDNA Synthesis Kit to prime at the beginning of the poly (A) tail.

• Choose the reverse transcriptase and DNA polymerase that can best transcribe and amplify the gene of interest.

Tip: Transcriptor High Fidelity cDNA Synthesis Kit can transcribe RNAs up to 14 kb and Expand 20 kb PLUS PCR System can amplify DNA up to 35 kb.

• Prevent mispriming by carefully designing your primers. Design alternative primers with a similar melting temperature and use both primers at the same concentration.

• Choose the appropriate DNA polymerase for a successful PCR outcome.

Tip: Select the best DNA polymerase for your application from our large enzyme selection.

• Increased enzyme concentrations can lead to decreased specificity and may not produce the desired result.

• Generally, lower Mg2+ concentrations produce more specific amplification, and higher concentrations produce more nonspecific amplification.

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What is important regarding dephosphorylation and ligation?

• For best results, ligate PCR fragments into the appropriate vector type. When a high protein expression level is the goal, choose the expression vector that is best for this application.

• Purify PCR fragments prior to using them in ligation experiments; because excess salts, nonspecific fragments, and primer-dimers can interfere with the ligation reaction.

Tip: The additional purification step immediately after restriction enzyme digestion can be avoided by using the Rapid DNA Dephos & Ligation Kit. Easily remove 5´ phosphates in 10 minutes and ligate in 5 minutes at room temperature using the kit's recombinant rAPid Alkaline Phosphatase. This excellent enzyme is quickly inactivated by heating to +75°C for 2 minutes. No time-consuming purification step is required.

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Which if any tag should be chosen?

The best epitope tag does not interfere with the function and cellular processing of the tagged protein, producing a strong signal in western blots and immunofluorescence microscopy. You cannot reliably predict how a particular tag will behave in a particular protein. For best results, consider the location of the tag within the protein (i.e., is it N- or C-terminal? Where is it within the reading frame?).

Note: Placing the tag at the C-terminus leads to the complete translation of the tagged protein; however, functional expression is dependent on tag location.

• Location of the tag at the N- or C-terminus can result in different yields of expressed protein.

• The histidine tag is not the first choice for mammalian cells; mammalian cells often contain proteins with runs/repeats of histidine, or endogenous myc protein, whereas the baseline level of these in insect cells is unknown. For mammalian cells, the following tags are thus preferred: HA, VSV-G, or Protein C.

Tip: Choose the Anti-HA High Affinity (rat monoclonal antibody clone 3F10) for the detection of hemagglutinin (HA)-tagged proteins in western blots and immunoprecipitation. Detect low amounts of protein and achieve high sensitivity. Minimize background due to lower cross-reactivity using reduced working concentrations compared to the Anti-HA clone 12CA5.

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Does the vector have influence on the protein expression yield?

Selecting a vector appropriate for your application is critical. When cells do not grow well after transfection, one may believe the heterologous protein is having deleterious effects on the cells, or that the transfection reagent is at fault. This may not be the case. Some vectors contain toxic sequences that cannot be recognized without prior testing. Moreover, experiments show that gene expression does not always correlate with cell growth. Cell proliferation may also not correlate with protein expression or the used transfection reagent, but may be a function of elements found within the vector itself. Gene expression profiling has been used to examine the effects of the vector backbone [1-3], further emphasizing the importance of vector selection appropriate to the application at hand.

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References

• [1] FuGENE® HD Transfection Reagent: Choice of Transfection Reagent with Minimal Off-Target Effect as Analyzed by Microarray Transcriptional Profiling. Susan Calvin, Jay Wang, Jeff Emch, Simone Pitz, and Linda Jacobsen. Biochemica 4, 2006.

• [2] Outstanding Microarray Experimental Results Using FuGENE® HD Transfection Reagent. Linda Jacobsen, Susan Calvin, and Simone Pitz. Biochemica 2, 2009.

• [3] Transcriptional Effects Of Transfection: The Potential For Misinterpretation Of Gene Expression Data Generated From Transiently Transfected Cells. Linda Jacobsen, Susan A. Calvin, and Edward K. Lobenhoefer. BioTechniques 2009 July, 47(1):617-24.

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