Gene Technology

Producing DNA fragments via gene technology

Identifying and isolating a specific gene that is a few hundred bases in length amongst millions of bases in eukaryotic DNA is quite challenging. There are three main techniques that scientists use to create DNA fragments:

1. Using reverse transcriptase to convert mRNA (messenger RNA) to cDNA (complementary DNA).
2. Using restriction endonuclease to cut the DNA molecule at a specific sequence.
3. Using a gene machine to create a gene-based on a protein with a known structure and composition.

Reverse transcription

This process uses a particular enzyme called reverse transcriptase. This enzyme naturally occurs in retroviruses such as HIV, and it creates DNA strands based on mRNA molecules.

• mRNA in cells correspond to the genetic sequence, and ribosomes read them to synthesise polypeptide molecules (proteins). This process is known as translation.
• A cell that naturally produces a protein should contain large amounts of that protein's mRNA, which can be isolated. For example, the beta cells in the islets of Langerhans in the pancreas secrete insulin and should contain high levels of insulin mRNA. So, the insulin mRNA can be extracted from beta cells.
• Following extraction of the desired mRNA, they can be treated with the reverse transcriptase enzyme, which polymerises deoxyribonucleotides and creates single-stranded cDNA molecules that have a complementary base sequence to that of the mRNA.

• The obtained cDNA molecules are subsequently treated with DNA polymerase in the polymerase chain reaction (PCR) process. The DNA polymerase synthesises a complementary DNA strand based on the cDNA, creating a double-stranded DNA. It also replicates the DNA molecules in repeated cycles that amplifies the number of DNA fragments.
• The main advantage of the reverse transcription process in isolating genes is that the product created at the end is a cDNA that does not contain any non-coding DNA (introns). This is important for inserting genes into prokaryotes since prokaryotic systems cannot remove introns.

Restriction endonucleases

Restriction endonucleases (RE) are enzymes that are naturally part of the bacteria's defence mechanism. They act by cutting the foreign DNA molecules.

There are various types of REs with active sites complementary to specific bases nucleotide sequences. These sequences are called recognitions sites, since each RE creates incisions in the DNA specifically at these locations.

We can classify REs into two main groups based on how they cut the DNA:

1. REs that incise the DNA at the exact location on both strands creates two blunt ends.
2. REs that cut both DNA strands at positions a few bases apart from each other lead to the creation of staggered ends with exposed DNA bases (bases without complementary pairs). These unpaired DNA bases can join to a DNA with complementary bases. These ends are also called sticky ends since they can easily join other DNA samples with sticky ends. The recognition sites for these types of REs have a palindromic sequence, meaning they are read the same on the forwards and reverse strands.

Fig. 3 - Creating DNA fragments with sticky ends using restriction enzymes

The gene machine

The gene machine is the most modern technique where scientists create DNA fragments in a laboratory using computers and special devices.

They first identify the protein of interest and examine its constituting amino acid sequence. Then, scientists can determine the mRNA and DNA sequences that could encode that protein by using the genetic code in reverse.

The obtained DNA sequence can then be entered into the computer. The computer checks the DNA fragments for biosafety and biosecurity, ensuring that it follows the various ethical regulations and does not violate international regulations. The computer then creates a series of small single-stranded nucleotides with overlapping sequences in an automated process. These strands are called oligonucleotides and can be assembled to generate the DNA sequence of the desired gene. Lastly, the engineered DNA strand is amplified and converted to the double-stranded DNA molecule using PCR.

The gene machine technique is accurate and can be conducted in as little as ten days. It also creates DNA strands that do not contain any non-coding regions (introns) and can be transcribed and translated by prokaryotic systems.

Table 1. Advantages and disadvantages of different gene technologies summarised.