Producing Fragments

Test your knowledge with free interactive questions on Seneca — used by over 10 million students.

Reverse Transcriptase

The target gene for producing recombinant DNA is obtained by producing DNA fragments. DNA fragments can be produced using reverse transcriptase.

mRNA

mRNA is a single-stranded molecule that is produced when a specific length of DNA (the target gene) is transcribed.
mRNA is complementary to the base sequence in the target gene.
The mRNA sequence can be used as a template for producing fragments of DNA.

Reverse transcriptase

Reverse transcriptase is an enzyme that converts single-stranded mRNA into double-stranded DNA.
Reverse transcriptase can be used in producing DNA fragments by converting the mRNA for the target gene into double-stranded DNA.
The DNA produced from reverse transcriptase is called complementary DNA (cDNA).

E.g. Insulin

DNA fragments of insulin are isolated from pancreatic cells in the following steps:
- mRNA for insulin is isolated from the pancreatic cells.
- The mRNA is mixed with reverse transcriptase.
- Reverse transcriptase converts mRNA into cDNA.
- The cDNA can now be used to produce recombinant DNA.

Restriction Endonucleases

The target gene for producing recombinant DNA is obtained by producing DNA fragments. DNA fragments can be produced using restriction endonuclease enzymes.

Recognition sequences

Recognition sequences are sections of DNA where the base sequence has palindromic base pairs.
- Palindromic base pairs have a sequence of base pairs that are the same but in opposite directions.
Recognition sequences can be used to isolate the target gene if there are two sets of sequences either side of the gene.

Restriction endonucleases

Enzymes called restriction endonucleases bind to recognition sequences.
Each restriction endonuclease binds to a specific recognition sequence (e.g. Eco RI is a restriction endonuclease that binds to GAATTC).
If two restriction endonucleases bind to two recognition sequences surrounding a target gene, the target gene can be cut out of the DNA.

Producing the fragment

DNA fragments can be produced in this way using restriction endonucleases. The steps involved are -
- DNA containing the target gene is mixed with the restriction endonucleases.
- Restriction endonucleases bind to the recognition sequences on either side of the target gene.
- The target gene is cut out of the DNA.

Gene Machines

The target gene for producing recombinant DNA is obtained by producing DNA fragments. DNA fragments can be produced using gene machines.

Synthesising DNA

DNA fragments can be produced by synthesising the target gene sequence using free-floating nucleotides.
This is useful because a DNA template isn't necessary.
The sequence for the target gene is obtained from a database.

Producing the fragment

DNA fragments can be produced in this way in the following steps -
- The sequence for the target gene is obtained from a database.
- Nucleotides are added in the correct order to synthesise the correct base sequence.
- Protecting groups are added throughout the synthesis to make sure the correct nucleotides are added and no side branches are produced.