Utilization of Type IIS Endonucleases in Molecular Cloning

Figure 2:  Identical fragments digested with Type IIS endonucleases are not compatible. A. The overhangs resulting from digestion with orthodox endonucleases are symmetrical and compatible. B. Overhangs resulting from digestion with Type IIS endonucleases are usually not. These overhangs are not compatible and would not be ligated to each other

Since orthodox Type II recognition sequences of the four different kinds of overhangs are symmetrical, their overhangs exhibit symmetry too. Therefore, the ends digested by a specific standard Type II enzyme are compatible with its identical fragments (Figure 2A). In this case, an EcoRI-HindIII fragment A isolated using an agarose gel is designed to ligate a HindIII-EcoRI fragment B; only a small portion of ligations are between a single fragment A and single fragment B. Most ligations are between two identical fragments as or two fragment Bs, subsequent ligations result in a few very large fragments. As a result, most isolated fragments are wasted insteadforming a circle of DNA.

In contrast, the sticky ends created by Type IIS endonucleases are usually not symmetrical. The overhangs resulting from digestion with BsaI usually would not be not be able to ligate the identical 5'-ATCG overhang, because they are neither symmetrical nor compatible as shown in Figure 2B.

This BsaI end only can ligate with a fragment with the 5'-CGAT overhang created by BsaI or another Type IIS endonuclease such as BbsI, BsmBI, and so on.

For example,

G G T C T C N /A T C G N N N

C C A G A G N T A G C/ N N N (Fragment C)

digested with BsaI, results in:

5'-A T C G N N N

N N N (Fragment C digestion)

The overhang would not ligate with its identical overhang (Figure 2).

In the case of fragment D

5'-/C G A T N G A G A C C

3'- G C T A/ N C T C T G G (Fragment D)

digested with BsmBI, results in:

5'- C G A T N G A G A C C

N C T C T G G (Fragment D digestion)

Ligation between fragments C and D would not result in any head-to-head or tail-to-tail connection (if the other ends are not compatible). Therefore, ligations between two asymmetrical ends created by (the same or different) Type IIS endonucleases would not result in waste. Highly efficient ligation between two asymmetrical sticky-ends is very useful in molecular cloning. The general applications of Type IIS endonucleases are summarized below.

i. Ligation among multiple fragments

The efficiency of ligation between three fragments digested by orthodox endonucleases is low. Ligation between four is almost impossible to select for a correct connection, because most fragments ligate to the wrong one (Figure 2A). Since an asymmetrical overhang has to ligate to another, different asymmetrical overhang, the ligation efficiency between them can be almost 100% if the two fragments are equal in mole. Therefore, it would allow successful ligation among multiple fragments [8,9].

ii. Creation of a large library due to high efficiency of ligation

A fragment can be isolated by digestion, with one or two different Type IIS endonucleases creating two different asymmetrical overhangs. The two different overhangs can result from a single Type IIS endonuclease, because the Type IIS endonucleases cleavage sequences are outside their recognition sequence. These fragments are not ligated unless there are other fragments that contain two ends compatible with the first fragment. Since the second fragment would also not ligate itself, the product of ligation only can be between the two ones. Therefore, the ligation efficiency in this scenario would be greater than the one between fragments isolated by digestion with orthodox enzymes. It is possible to make a large plasmid library (>10⁶) by using Type IIS endonucleases and electroporation technology (Zhang, unpublished data).

iii. Seamless connection of two sequences

Traditional cloning requires a connection between two fragment digestions by two enzymes with compatible ends. In most cases, if there is not a restriction site, it would be very easy to design a PCR primer containing a restriction site within the primer. However, if a project does not allow us to mutate the sequence, it would be not difficult to design a primer that contains a Type IIS restriction site. As mentioned above, the ATCG/CGTA created by BsaI can be replaced to any four nucleotide overhangs, making it possible to seamlessly connect any two sequences without the insertion or deletion of a single base [10].

iv. Creating an overhang compatible with ends digested by orthodox endonucleases

In case a cloning strategy is planned, a fragment is needed to insert between the EcoRI and HindIII sites within a plasmid vector; the fragment can be amplified by PCR with two primers. The first one contains an EcoRI site, while the second one contains a HindIII site. The PCR product then can be digested with the two endonucleases and the EcoRI-HindIII fragments can be used for insertion. However,attention must be paid to whether this fragment contains other EcoRI or HindIII sites, or the result would be in the digestion of this fragment into smaller pieces, making the product of ligation contain deletion (s). If there are more than one EcoRI or HindIII sites within this fragment, the first step can be checking whether there are EcoRI or HindIII compatible sites. The MfeI site (CAATTC) is compatible with the EcoRI site (GAATTC); both enzymes create the 5'- AATT overhang. No enzymes however, are compatible with HindIII (AAGCTT). If this fragment contains additional HindIII site (s), a 4-base 5' overhang of Type IIS endonucleases can be used to create the 5' overhang. There are at least three Type IIS endonucleases that recognize 6 bases (Table 1); in most cases, at least one from the three enzyme recognition sites would not be within this fragment. It is possible to design a primer that contains one of the three Type IIS sites. The digestion sites can be designed as 5'-AGCT (compatible with HindIII digestion) and the recognition sites can be designed outside the digested fragment. The fragment can be digested with EcoRI and the Type IIS endonuclease (instead of HindIII) and is used to insert between the EcoRI and HindIII sites of the plasmid. In addition, a choice can be made if the ligation site can be re-digested with HindIII by adding an A before the AGCT overhangs. If it is preferred that the ligation site not be digested with HindIII, the A can be replaced with T, C or G. Many Type IIS endonucleases are commercially available now. There are 5 possibilities resulting from digestion with an orthodox restriction enzymes described above (Figure 1). Commercially available Type IIS endonucleases can create the 4 possible overhangs resulting from any orthodox endonucleases except the 3' 4 base overhang. BstXI is not a Type IIS but Type IIP endonuclease. Digestion with BstXI results in a 3' 4 base overhang; meanwhile three bases have to be added to the PCR fragment, which probably can serve most purposes.

  

    
3' end 2    base overhang:
    
BsrDI
    
G  C     A  A  T     G   N  N /
      
C  G     T  T  A  C    / N  N
  
  

    
 
    
BtsI
    
G  C     A  G  T     G   N  N /
      
C  G     T  C  A  C    / N  N
  
  

    
5' end 2    base overhang:
    
FauI
    
C  C     C  G  C     N  N  N  N    / N  N
      
G  G  G  C     G  N  N     N  N   N  N    /
  
  

    
5' end 3    base overhang:
    
BspQI
    
G  C     T  C  T     T  C  N / N     N  N
      
C  G     A  G  A     A  G  N      N  N  N /
  
  

    
 
    
EarI
    
C  T     C  T  T     C  N / N  N  N
      
G  A     G  A  A     G  N   N     N  N /
  
  

    
5' end 4    base overhang:
    
BsaI
    
G  G     T  C  T     C  N / N  N  N  N
      
C  C     A  G  A     G  N   N     N  N  N /
  
  

    
 
    
BsmBI
    
C  G     T  C  T     C  N / N  N     N  N
      
G  C     A  G  A     G  N   N     N  N  N /
  
  

    
 
    
BbsI
    
G  A     A  G  A     C  N  N / N     N  N  N
      
C  T     T  C  T     G  N  N      N  N  N  N    /
  
  

    
blunt
    
MlyI
    
G  A     G  T  C     N  N  N     N  N /
      
C  T     C  A  G     N  N  N     N  N /
  
  

    
3' end 4    base overhang:
    
BstXI*
    
C  C     A  N / N  N     N  N   N     T  G  G
      
G  G     T  N   N     N  N  N / N     A  C  C
  

Table 1:   Most useful Type IIS endonucleases.