Restriction Endonucleases

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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 antiparallel base pairs.
- Antiparallel 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.

Reverse transcriptase

Rather than cutting out the gene with restriction endonucleases, reverse transcriptase can be used in an alternative method.
Reverse transcriptase can convert the mRNA of the desired gene into DNA.
- The mRNA can be extracted from target cells.

In vivo

After DNA fragments have been produced, they can be amplified either in vivo (inside the organism) or in vitro (outside the organism). The steps involved for in vivo amplification are:

1) Forming sticky ends

A vector is a form of transport for the DNA fragment.
Vector DNA is cut open by enzymes called restriction endonucleases. The enzymes cut the DNA at a specific region called recognition sequences.
Restriction endonucleases cut the vector DNA so that each end has a short single-stranded section.
The ends of the DNA that are single-stranded are called the sticky ends.

2) Sticky ends on fragment DNA

The DNA fragments have sticky ends that are complementary to the sticky ends on the vector DNA.
This is because the DNA fragments have either been cut from DNA using the same restriction endonucleases or because several nucleotides have been added onto the ends of the fragment.

3) Inserting into vector DNA

The sticky ends on the DNA fragment and vector DNA bind together.
An enzyme called DNA ligase attaches the sticky ends together. This is called ligation.
The DNA fragment has been inserted into the vector DNA. This is recombinant DNA.
The DNA fragment needs to contain promoter sequences to ensure transcription.

4) Transferring to host cells

The vector transfers the recombinant DNA to the host cells.
If the vector is a plasmid (small, circular DNA found in bacteria) -
- The host cells take up the recombinant DNA via heat-shock. This is where the cells are heated at 42°C for one minute.
If the vector is a bacteriophage (virus) -
- The recombinant DNA is injected into host cells.

5) Inserting marker genes

The cells that have successfully taken up the recombinant DNA are transformed. Transformed cells are also said to be genetically modified (GM).
Not all the cells will be transformed.
The transformed cells are identified using marker genes.
Marker genes are genes that are inserted along with the recombinant DNA and confer antibiotic resistance.

6) Identifying transformed cells

Transformed cells can be identified by placing the cells on an agar plate with antibiotics.
Only cells that have successfully taken up the recombinant DNA will be able to survive on the antibiotic agar plates.
Transformed cells can then be grown in large numbers to amplify the target gene.
Sometimes fluorescent genes or genes for easily dyed substances are also used as marker genes.

In vitro

In vitro amplification uses the polymerase chain reaction (PCR). PCR can rapidly increase the number of copies of DNA fragments. The steps involved for in vitro amplification are:

1) Set up the reaction mixture

The DNA fragments are mixed with -
- Primers (short sections of DNA).
- An enzyme called DNA polymerase (produces new strands of DNA).
- Free-floating nucleotides.
Together these components form the reaction mixture.

2) Heat to 95°C

Heat the reaction mixture to 95°C.
The high heat causes the hydrogen bonds between DNA strands to break and the DNA to separate into two separate strands.

3) Cool to 65°C

Cool the reaction mixture to 65°C.
This causes the primer to anneal to the two separate strands of DNA.
The primers are complementary to the beginning of the two strands.

4) Heat to 72°C

Heat the reaction mixture to 72°C.
- This is the optimum temperature for Taq polymerase activity.
- Taq polymerase is a DNA polymerase enzyme from organisms that grow at high temperatures.
Taq polymerase produces two new strands of DNA by using the two separated strands of DNA as a template.
Taq polymerase adds free-floating nucleotides that are complementary to the template strands of DNA.
Primers allow the nucleotides to bind to one another and produce a strand of DNA.

5) Repeat

This process of heating, cooling and heating produces two new strands of DNA from one strand.
The process can repeated as many times as possible to quickly amplify the number of DNA fragments.
The number of DNA fragments is doubled in each cycle of PCR.