Enrichment of Oligonucleotide Directed Mutagenesis with M13 Using Kunkel Method

Share This Post:

In previous post, “Oligonucleotide Directed Mutagenesis with M13 DNA as a Template” was discussed. In this post, how to enrich desired mutation containing strand in order to achieve approximately 100% modified strand with desired mutation as compared to wild type strand.

What would be the outcome of this post?

In this post, students, general audience will be able to know how to enrich mutant gene inserted in M13 cloning by using Kunkel method

In previous post some basic knowledge of M13 as a cloning vector was discussed. M13 can be used as a cloning vector to clone the inserted gene with specific mutation.

How is it possible to mutate the inserted gene in M13 ss DNA?

We can perform by few steps for example.

First insertion of gene of interest which we want to mutate using ss DNA of M13 as a template.
Then replication of the other strand to convert it into ds DNA using mutated oligonucleotide primer and Klenow fragment as Polymerase enzyme.
Then ligation of second strand with T4 DNA ligase and transformation to specific host.

Now host cell has two type of cloning vectors, one can amplify and express normal version of gene of interest, while other one with desired mutation. Each one is present with 50% chance.

Our previous post is about M13 DNA as a cloning vector. In case of M13 vector, inserted gene can be mutated using mismatched oligonucleotide primer. But the main drawback is that the resulted product of amplification will be 50 % with wild type DNA and 50% will be consisted of desired mutant version.

Here the question arises, “How to enrich our desired mutant gene? This is the main objective of this post to discuss the strategy of enriching mutant gene DNA quantity in order to minimize the contamination of wild type DNA.

Kunkel Method

Here is a summary of how to enrich mutated DNA with our desired mutation in M13 DNA. To this issue Kunkel methodcan be applied. In this method, first we introduce our desired mutation through mismatch oligonucleotides strand in gene of interest. In this method, either single base pairs or larger that can include insertions, deletions or substation mutations. Please follow PEP01 post to know these mutations.

Requirements of Kunkel Method

Before going to perform mutagenesis process, make sure the availability of these substances.

Two E. coli strains (wild and special types)
Klenow Fragment enzyme
M13 DNA as a cloning vector

Steps in Kunkel Method

Kunkel method is divided into three main steps; such as; first and last steps belong to ‘in vivo process’ while middle step is known as ‘in vitro process’. In this section, these three steps will be discussed in detail.

1. In vivo process

In this step a special type of E. coli strain is used. This strain lacks two enzymes that are involved in the replication process of DNA. In vivo process consisting of these steps.

Formation of Hybrid DNA molecule. A hybrid DNA molecule is constructed by inserting a target DNA fragment into M13 DNA. Target DNA means the DNA that is to be mutated.
Transfection of Hybrid DNA molecule into special strain of E. coli. This hybrid DNA, consisting of M13 DNA plus target DNA, is transfected into a special E. coli strain. this E. coli strain has some deficiency when compared with normal E. coli strain.
Deficiency in E. coli strain. For example, this strain lacks uracil deglycosidase and dUTPase. Due to deficiencies of these two enzymes, this strain is normally known as ung-dut-strain.

Functional role of these two enzymes

dUTPase is involved in the production of dUMP from dUTP. dUMP works as an immediate precursor of thymidine nucleotides. dUMP productin from dUTP leads to decrease the intracellular concentration of dUTP. Therefore, chances of incorporation of uracil instead of thymidine into DNA become reduce. Uracil is inserted into new synthesizing strand of DNA in the form of dUTP.

uracil-DNA glycosylases, main role in base-excision repair pathway, work as a repair system and involved in repairing mismatch nucleotide. Normal version of this enzyme inhibit mutagenesis in DNA. As the name of this enzyme show, uracil incorporated into DNA molecule is eliminated. This task this enzyme performs removing uracil nucleotide. N-glycosylic bond is responsible for the attachment of Uracil with pentose sugar. This enzyme is responsible to cleave the N-glycosylic bond between uracil and pentose sugar.

Normally DNA molecule consist of A, G, C and T nucleotide. There are two ways (Deamination & Mis-incorporation) to have uracil in DNA molecule. 1). Deamination is the process of removal of amin group from cytosine and converting cytosine into uracil 2). Mis-incorporation of dUMP residues.

Advantage of lacking these enzymes

What happened if dUTP is inserted into newly synthesized DNA strand? When uracil is incorporated into newly synthesized DNA, an enzyme, known as uracil deglycosidase, recognize and eliminate the mismatch nucleotide in DNA strand. The right nucleotide is again incorporated into new synthesized DNA strand.

What happened if dUTP is incorporated into newly synthesized DNA strand in a bacterium that lacks uracil deglycosidase?

dUTP is accumulated in bacteria in the absence of dUTPase enzyme. Therefore, dUTP concentration increases in cell that is incorporated into DNA strand synthesis in place of T nucleotide during replication process. However, mismatch nucleotide mechanism is present in bacteria which eliminate the incorporated wrong uracil nucleotide. Bacteria perform this function through uracil deglycosidase. When bacteria strain unable to synthesize uracil deglycodidase, then such type of bacteria unable to repair mismatch uracil nucleotide by eliminating from new DNA strand. This hybrid plasmid, containing Uracil instead of Thymidine, is transformed into bacteria.

2. In vitro process

In this steps mutant DNA is amplified by special type of enzyme, known as Klenow Fragment.

Detail. Hybrid plasmid containing target DNA, is placed with a synthetic oligo. This synthetic oligo match with target DNA but unable to make pair with the nucleotide located at the position of desired mutation. You can see here in this figure that all other nucleotides in green strand are matching with template strand except T (figure coming soon). The mixture is then preserved Klenow fragment that incorporate dNTP’s into newly synthesize fragment. Later DNA ligases are used to seal the gap or nick in the new synthesized strand. Now the M13 consists of ds DNA, one strand contains uracil nucleotide instead of thymidine while the other strand remains with normal Thymidine nucleotide.

Klenow fragment

Subtilisin, a protease enzyme, is used to cleave DNA polymerase 1 of E. coli into two fragments, one having large while the other fragment remains small after cleavage process. This large fragment is known as “Klenow Fragment”

Characteristics of Klenow Fragment

DNA polymerase 1, of E. coli, plays a dual role during DNA replication. It is responsible for polymerase activity using 5’→3′ direction during replication process. Simultaneously, it is also involved in exonuclease activity in 5’→3′ or 3’→5′ directions. This exonuclease activity is necessary for mismatch repair during DNA replication.

Once DNA polymerase 1 is treated with the subtilisin to become fragments of large and small size. Polymerase and exonuclease activities of DNA polymerase1 is adversely disturbed. For example, the large fragment, known as Klenow fragment, maintains polymerase activity as well as exonuclease activity in the direction of 5’→3′ and 3’→5′ respectively. Polymerase activity is necessary for DNA new strand synthesis while exonuclease activity is responsible for proof reading. However due to the absence of small fragment, Klenow fragments is unable to perform exonuclease activity in 5’→3′.

On the other side, small fragment is unable to perform polymerase activity as well as exonuclease activity in the direction of 5’→3′ and 3’→5′ respectively. Small fragment remains only perform 5’→3′ exonuclease activity.

Application of Klenow Fragment

Klenow fragment has wide applications in molecular biology research. Such as Klenow fragment is used to synthesized complementary strand of cDNA. In addition,

Sanger sequencing method can also perform by using Klenow fragment.
Due to their exonuclease activity, we can use Klenow fragment to produce blunt ends of sticky end containing DNA molecule. Sticky end may be at 5´ or 3´ overhangs.
- Klenow fragment can fill-in 5´ overhangs consequently the sticky end become blunt ends. In parallel to filling 5´ overhangs, it has the ability to chop up 3´ overhangs that leads to form blunt ends
Klenow fragment is also used in preparation of radioactive DNA probes.

3. In vio process

As DNA is amplified, this DNA is transferred into wild type of E. coli.

Details. Now M13 DNA is present in hybrid form (consisting of wild strand and U nucleotide containing strand). This hybrid DNA is transformed into normal E. coli. Hence this strain is wild type and both enzymes, which lacks special strand, are present. Therefore, Base repairing system of bacteria detect unnatural strand and starts degrading DNA strand, containing uracil as well as to produce a strand containing T. For this purpose, the new mutagenized DNA strand work as template for amplification in bacteria. Now both strands contain the newly mutated sequence.

In this way M13 DNA can be used to enrich mutant strand in order to get desired change DNA for the desired synthesis of protein with desired characteristics in Protein Engineering.

Frequently Asked Questions and Answer

In 1985, Thomas Kunkel introduced a method to enrich the desired mutation-containing fragment of DNA of the desired gene. Three steps are required to execute site directed mutagenesis using the Kunkel method.

Briefly, these steps are;

1). In vivo step. A desired gene fragment of DNA is inserted into a ssDNA vector (such as M13 etc.). The aim of this step is to convert ssDNA vector into the dsDNA vector along with the template DNA strand. ssDNA vector containing vector is transformed into a special type of bacteria strain (E. coli dut^–ung^–) to convert template DNA containing ssDNA vector into ds DNA molecule. Due to enzymes deficiencies (i.e., dUTPase and Uracil deglycosidase), the other strand is synthesized to consist of deoxy Uracil nucleotides (dU) instead of deoxy thymidine (dT) nucleotides.

2). In vitro step. First of all, the double stranded hybrid vector is converted into a single stranded form. Now the dU containing ss DNA vector along with the template strand is subjected to incubation with desired mutation encoding-primer (synthesized against the dU-strand of vector) and Klenow fragment. The Klenow fragment synthesizes the other strand against the ss-dU containing strand. At the end of this step, the dsDNA vector, consisting of a dU-containing strand and a normal strand along with the desired mutation, is synthesized.

3). In vivo step. Vector, synthesised in step 2, is transformed into normal bacteria (E.coli). The E. coli DNA repair system degrades the dU-containing strand and replicates the other stand. The other stand consists of the desired mutation. Now both strands consist of the desired mutation. Further amplification of this vector propagates the desired mutation.

Hence, using the Kunkel method, 100% desired mutation-containing vector can be synthesized.

Klenow fragment is large part of the DNA polymerase after cleavage process using subtilisin. This fragment has the polymerase activity but lacking exonuclease activity. Therefore, DNA strand can be synthesized without performing mismatch repair. Consequently, the other dU-containing strand remain mismatch during the process of replication using Klenow fragment.