Available cloning methods

SeqBuilder Pro supports the following cloning methods:

Gibson (LIC, SLIC, CPEC, SLiCE) Cloning

Gibson assembly and Ligation Independent Cloning (LIC) are techniques offered by New England Biolabs. Both cloning methods can be simulated in SeqBuilder Pro using the five-step cloning workflow. Note that Gibson cloning allows up to 6 fragment inserts. When using this cloning method, restriction insert size must be relatively large in order to prevent primer overlaps that could keep the fragment from being incorporated into the expression clone.

In-Fusion Cloning

In-Fusion cloning is a technique offered by Clontech/Takara, and allows a maximum of 2 fragment inserts. This cloning method can be simulated in SeqBuilder Pro using the five-step cloning workflow.

GeneArt Cloning

GeneArt cloning is offered by ThermoFisher Scientific and allows up to 4 inserts. This cloning method can be simulated in SeqBuilder Pro using the five-step cloning workflow.

Golden Gate Cloning

Golden Gate Cloning (GGC) is a molecular biology technique developed by New England Biolabs that allows for the efficient and precise assembly of multiple DNA fragments. By utilizing Type IIS restriction enzymes, which cleave DNA outside of their recognition sequence, this method enables the creation of complex genetic constructs in a single reaction. Unlike traditional cloning methods, GGC avoids the generation of unwanted DNA fragments, resulting in high cloning efficiency and reduced background. This method is particularly useful for creating complex genetic constructs and libraries, making it a useful tool for synthetic biology, metabolic engineering, and gene therapy.

Golden Gate Cloning has gained popularity in recent years because it offers several advantages to most other methods:

• Seamless/scarless cloning of multiple inserts — Other methods leave behind scars or spacers: pieces of the cloning methodology that can’t be removed using the technology involved. GGC allows the selection of the overhang to be taken from anywhere around the beginning of the inserts, leading to a near-perfect rejection of the other overhangs in the set. Because this might lead to skipping some of the beginning of the insert, the skipped part is added to the end of the preceding insert/vector, meaning nothing is lost after assembly.

• Large capacity — This method supports directional orientation of 2-50+ inserts.

• Simplicity — A single test tube is used to simultaneously perform the digestion and ligation steps.

Golden Gate cloning differs from other methods in that it uses exactly one enzyme to cut all the parts. Golden Gate enzymes are Type IIs cutters that have three or four base overhangs and cut outside their recognition sequences. As a result, the GGC method creates primers containing the recognition sequence and overhangs designed to fit together in an organized way without risk of mis-ligating (i.e., placing the components of the clone in the wrong order).

The process of designing a Golden Gate clone involves several steps.

1. Design primers that make overhangs and cut sites for all the inserts.

2. Cut to remove the enzyme and leave the overhang.

3. Ligate to match up the overhangs that have been carefully chosen to be unique.

Amplification, cutting, and ligating usually (though not always) takes place in the same tube. Having the recognition sequences cut apart and absent from the assembled product allows the cutting and ligation to occur simultaneously.

When using the GGC method within SeqBuilder Pro, an added bonus is that features are created automatically for all components of the clone. For step-by-step instructions to performing virtual GGC in SeqBuilder Pro, we recommend following our Golden Gate Cloning tutorial rather than the generalized “Step (number)” subtopics under Automated Virtual Cloning. The tutorial contains helpful troubleshooting information for correcting a clone with issues.

For more information on GGC in general, see this page at the NEB website.

MultiSite Gateway Pro Cloning

MultiSite Gateway Pro technology enhances the classic Gateway cloning protocol by allowing you to clone up to four regions at one time. In MultiSite Gateway Pro cloning, three additional types of recombination sites have been engineered to recombine uniquely between the two ends of a source sequence and a host vector. MultiSite Gateway Pro cloning can be simulated in SeqBuilder Pro using the five-step cloning workflow.

The recombination features used in MultiSite Gateway cloning are as follows:

• For Expression Clones: attB1, attB2, attB3, attB4, and attB5

• For Donor Vectors: attP1, attP2, attP3, attP4, and attP5

• For Entry Clones: attL1, attL2, attL3, attL4, and attL5

• For Destination Vectors: attR1, attR2, attR3, attR4, and attR5

*Note: When an r is added to any of these feature names, that indicates the reverse of the specified site. For example, L5r indicates an L5 site in reverse.

As in classic Gateway cloning, attL and attR sites recombine, and attB and attP sites recombine. The recombined sites transform by swapping opposite halves into the opposite set, so attL and attR become attB and attP and vice versa.

Click here for information on the specific 2, 3, and 4 fragment MultiSite Gateway protocol from Invitrogen.

For detailed information on creating a suitable vector, see the section “Preparation of Gateway cloning vectors” in Add custom vectors to the Cloning Vector Catalog.

*Note: During the cloning simulation, primers are altered to match the donor vector in the first clone. When creating inserts with attB1-attB2 primers, SeqBuilder Pro automatically adds four guanines (GGGG) to the 5’ end of the primer, as per the requirement specified in in Thermofisher’s online Gateway Technology Manual.

Gateway Cloning

Gateway cloning is based upon the site specific recombination of lambda bacteriophage. Two types of recombination sites have been engineered to recombine uniquely between the two ends of a source sequence and a host vector. The sites have also been engineered to allow the recombination reactions to be completely reversible using two sets of catalytic proteins. These two reactions are called LR and BP, so named because of the recombination sites involved in each; LR recombines attL and attR sites, BP recombines attB and attP sites. The recombined sites transform by swapping opposite halves into the opposite set, so attL and attR become attB and attP and vice versa. Vectors engineered to contain these sites have been given the names: Entry Clone (attB), Expression Clone (attL), Donor Vector (attP), and Destination Vector (attR).

When you perform a Gateway cloning simulation in SeqBuilder Pro, you first create an Entry Clone for a specified fragment insert. This Entry Clone (rather than a fragment insert) is then used as input for making an Expression Clone. Both type of clones are created using the cloning workflow. You will follow the workflow once to create the Entry Clone, then again to create the Expression Clone.

A special type of Gateway cloning is RNA interference (RNAi) cloning. This procedure uses Gateway recombination technology and a special vector (pHELLSGATE and its derivatives) containing two pairs of recombination sites opposing each other on opposite strands. The cloning system is intended to create an RNA transcript under expression that interferes with the translation of the cloned gene, thereby suppressing it.

• To construct inverted repeat Entry Clones, use the vectors pHELLSGATE and pHELLSGATE 4.

• To construct inverted repeat Expression Clones from the single site Entry Clones, use the vectors pHELLSGATE 8 and pHELLSGATE 12.

For detailed information on creating a suitable vector, see the section “Preparation of Gateway cloning vectors” in Add custom vectors to the Cloning Vector Catalog.

PCR-Directed Restriction Cloning

PCR-directed restriction cloning utilizes the unique activity of site-specific restriction enzymes. First use restriction enzyme cut sites to create the insert. If no convenient restriction sites are available, you may instead select the region you want to clone and SeqBuilder Pro will engineer primers to create sites compatible with the selected sites of the chosen vector. In either case, proceed to follow Steps 2-5 of the cloning workflow.

Using a cloning vector with compatible restriction sites, the insert and vector can be digested with restriction enzymes to generate complementary ends, thereby enabling the vector and insert to be efficiently ligated. SeqBuilder Pro uses the basic idea very similar to the copy/paste of text editing. The primary difference in SeqBuilder Pro is the way the sequence endpoints are selected and what happens after the paste command. The cloning workflow simplifies this further by making the selection of restriction sites easier and by eliminating the need to find matching sites in the cloned region.

TA Cloning

TA Cloning is a cloning method that takes advantage of the terminal transferase activity of some DNA Polymerases that add a 3’-A overhang to each end of a PCR product. A linearized vector with prepared 3’-T overhangs facilitates ligation of the PCR product. TA cloning can be simulated in SeqBuilder Pro using the five-step cloning workflow. In Step 1, follow the instructions for Option E – Using the PCR product of the current primer pair as the insert.

Directional TOPO Cloning

Directional TOPO cloning utilizes the binding activity of Topoisomerase I, and can be simulated in SeqBuilder Pro using the five-step cloning workflow. A sequence of interest is PCR amplified using a forward strand primer with four additional bases (CACC). Commercial vector sequences have been engineered with topoisomerase bound at 3’ and a complementary 5’ GTGG overhang. The overhang invades the 5’ end of the PCR product and anneals to the added bases. The 5’ hydroxyl group of the PCR product attacks the topoisomerase bound to the vector, releasing it and causing the ligation of the phosphate backbone. Orientation of the insert is determined by the 5’ overhang, allowing the other end to be blunt.