What are attB sites?
The attB site is a short DNA sequence (less than 30 bp) corresponding to the crossover region at which strand exchange takes place (7). Two imperfect inverted repeats that bind to the integrase surround a 7-bp overlap region delimited by the scattered cuts made by the recombinase.
How does BP Clonase work?
The BP clonase enzymes recombine the attB and attP sites, replacing the Gateway cassette with the amplified insert, which is now flanked by attL or attR sites depending on the configuration of the DNA fragments and vectors.
What is Clonase?
Gateway BP Clonase Enzyme Mixes Gateway BP Clonase enzyme contains both Int (Integrase) and IHF (Integration Host Factor) proteins that catalyze the in vitro recombination of PCR products or DNA segments from clones (containing attB sites) and a Donor vector (containing attP sites) to generate Entry clones.
What is BP and LR reaction?
The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone with all of the components necessary for gene expression. The LR Reaction takes place between the attL sites of the generated entry clone and the attR sites of the destination vector.
What is LR reaction?
The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. The entry clone generated from the BP reaction includes the attL sites. The Destination vector is designed to include the attR sites.
What is a TOPO cloning vector?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
What is attB1?
att: It stands for attachment site generally present in phage viruses and bacteria. attB: Stands for bacterial attachment site which helps in site specific recombination and always attaches with the PCR product. It consist of four homologous core sequence of ~25 bp each, attB1 ACAAGTTTGTACAAAAAAGCAGGCT.
What is the purpose of TOPO TA cloning?
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.
What is TOPO cloning used for?
What is TOPO reaction?
Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the basic ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds.
What is TA in genetics?
“TA” is short for “thymine” and “adenine.” This cloning technique utilizes the ability of thymine to hybridize to adenine in the presence of ligases. Restriction enzymes are not used, unlike the traditional subcloning method. Instead, PCR products are amplified using Taq polymerases enzymes.
How are the gateway attb1 and attb2 sequences added to a gene?
1) “Gateway attB1, and attB2” sequences are added to the 5’, and 3’ end of a gene fragment, respectively, using gene specific PCR primers and PCR-amplification;
What is the difference between attb1 and attb2 and attl2?
They have modified versions of the attB, P, L and R sites that recombine very specifically and directionally: attB1 sites react only with attP1 sites; attB2 only with attP2, attL1 only with attR1; attL2 only with attR2, and so on.
What are the att sequences used for?
These are useful for stitching together entry and destination sequences to calculate the sequence of a final expression clone. Below are all of the att sequences (attB, P, L, and R) used in the original three-part multisite Gateway system.
What is the difference between attP and attB sites and Gateway vectors?
Recombination of attP and attB sites creates attL and attR sites. Gateway vectors contain modified versions of the att sites so that scientists can easily clone in their desired DNA sequences. Gateway technology relies on the two reactions described below: