Performing a restriction digest in the laboratory
Digest a sample of λ DNA (concentration 125 μg ml−1) with HindIII
- DNA – The amount of DNA required for restriction digestion varies depending on the experiment and its desired results. – To digest 2 μg of λ DNA -16 μl of the DNA sample is needed
- Buffer solution – used to adjust the pH and ionic strength of the solution containing DNA prior to adding the endonuclease. Ionic strength is usually provided by sodium chloride, and magnesium. Reducing agent such as dithiothreitol (DTT) is also added to the buffer solution. It stabilizes the enzyme and prevents its inactivation.
- Restriction endonuclease
- Incubation temperature – most restriction enzymes functions at 370C. After one hour of incubation reaction is almost completed and enzymes should be inactivated. By terminate the reaction (destroy the enzyme), short incubation at 70 ◦C, a phenol extraction, the addition of EDTA, which binds Mg2+ ions preventing restriction endonuclease action
Analysing the result of restriction endonuclease cleavage
- DNA molecules are negative charges, placed in an electric field will migrate towards the positive pole.
- Separation of molecules by gel electrophoresis – During agarose gel electrophoresis, a DNA fragment will migrate at a rate that is proportional to its size
- Gel electrophoresis is used to stain the gel that makes the DNA visible.
Ligation: Joining DNA molecules together
The DNA molecule is created when the vector molecule and the DNA to be cloned are joined together.
Sticky ends increase the efficiency of ligation – Blunt ends are more challenging to ligate efficiently than sticky ends because they lack single-stranded regions that can help facilitate the pairing of DNA molecules, making it harder for the ligase enzyme to form a covalent bond between them. In contrast, sticky ends increase the likelihood of successful ligation due to their complementary base pairs which enable accurate alignment and binding.
Sometimes vector molecules has sticky ends but target DNA fragments has blunt end. Give sticky ends to the DNA fragments using Linkers, Adapters, Homopolymer tailing
Linkers
It is important to note that linkers can be designed with either blunt ended or sticky ended, usually blunt-ended containing specific restriction sites. Blunt-ended linkers are able to ligate DNA fragments, but the efficiency of this process is generally lower compared to when using compatible sticky ends. However, adding a restriction site within the linker allows for efficient ligation by creating compatible sticky ends through the use of restriction enzymes. Once ligated, the linker serves as a bridge for inserting DNA into cloning vectors.
Adaptors
Adaptors are short synthetic oligonucleotides that are designed to have one sticky end.
The purpose of adaptor is ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to produce a new molecule with sticky ends fragment, to produce a new molecule with sticky ends Ligation: Joining DNA molecules together fragment, to produce a new molecule with sticky fragment, to produce a new molecule with sticky
When adaptor molecules are synthesized the blunt end is the same as natural DNA, but the sticky end is different; 5′ -P terminus is modified: it lacks the phosphate group, and is in fact a 5′ -OH terminus
Homopolymer tailing
Homopolymer tailing is a technique used to create single-stranded overhangs on the ends of double-stranded DNA molecules. This process involves adding a homopolymer tail to blunt ends via terminal deoxynucleotidyl transferase (TdT) and a specific type of deoxynucleotide (dNTP).
Now they can be ligated with another molecule that has complementary overhangs.
Cloning PCR products
Cloning PCR products is a widely used technique for obtaining multiple copies of an amplified DNA fragment.
This process involves inserting the fragment into a cloning vector, such as a plasmid or phage vector, and then transforming it into a host organism like E. coli bacteria.
dT Vector cloning
PCR is an important technique used to amplify DNA. The Taq polymerase enzyme, which takes part in this process, adds an adenosine (A) at the end of each synthesized strand. As a result, the double-stranded product produced by PCR will have single nucleotide overhangs at its 3′ termini.
Cohesive-ended cloning
- Oligonucleotide primers can be designed with a restriction endonuclease site incorporated into them.
- After PCR amplification, the products are treated with the restriction enzyme to cut each molecule within the primer sequence and generate sticky-ended fragments
Uptake of DNA by bacterial cells
Preparation of competent E. coli cells –
- Prior treatment with Ca 2+ at 4°C
- A brief increase in temperature termed heat shock
- Small, circular molecules are taken up most efficiently than liners molecules