21- Recombinant DNA technology

What is recombinant DNA?

DNA of two different organisms that has been combined

What is an organism containing recombinant DNA known as?

• Transgenic

• Genetically modified organism (GMO)

Why is DNA from organisms accepted by organisms of different species?

Because the genetic code is universal (the same in all organisms)

What are the names of the stages involved in gene transfer and cloning?

• Isolation

• Insertion

• Transformation

• Identification

• Growth/ cloning

What are the three ways in which a gene can be isolated from the rest of the DNA sequence?

• Converting mRNA to cDNA using reverse transcriptase

• Use of restriction endonucleases

• Creating the gene in a gene machine

What is reverse transcriptase able to do?

Produce DNA from RNA (reversing transcription)

How are DNA fragments which code for the desired protein produced using reverse transcriptase? (5 marks)

• A cell that readily produces the protein is selected (e.g. β cells of the islets of Langerhans to produce insulin)

• These cells have large quantities of the relevant mRNA, which is therefore more easily extracted

• Reverse transcriptase is used to produce DNA from RNA

• This DNA is called complementary DNA, cDNA

• DNA polymerase builds up complementary nucleotides on cDNA template to produce double-stranded DNA fragment

What are palindromic sequences?

• Base sequences of sticky ends produced by restriction endonucleases

• Complementary to each other

How are DNA fragments coding for a desired protein produced using a gene machine? (7 marks)

• Work out amino acid sequence of desired protein, use this to work out mRNA codons, use these to work out DNA triplets

• Feed desired sequence of bases into computer

• Sequence is checked for biosafety and biosecurity (to ensure international standards and ethical requirements are met)

• Computer synthesises oligonucleotides

• In an automated process, oligonucleotides are assembled in the correct order using overlapping sections

• Gene is replicated using PCR

• Genes produced are checked using standard sequencing techniques and those with errors are rejected

What are oligonucleotides?

Small, overlapping single strands of nucleotides produced by computers

What are the advantages in using a gene machine to produce DNA fragments?

• Any sequence of nucleotides can be produced

• In a very short time

• With great accuracy

• Also, genes are free of introns so can be transcribed and translated by prokaryotic cells

What are the two general methods used to clone DNA fragments?

• In vivo cloning

• In vitro cloning

What does in vivo cloning mean?

Gene is cloned within living organisms

What are sticky ends?

Exposed nucleotide bases produced from restriction endonucleases

When will the sticky ends on different DNA fragments be complementary to each other?

When the DNA is cut using the same restriction endonuclease

How is a DNA fragment prepared for insertion into a vector?

• Promoter region is added

• Terminator region is added

Why are promoter and terminator regions added to DNA fragments before insertion into a vector?

So that RNA polymerase can start and stop transcription of the gene at the appropriate points

What is the most commonly used vector that a DNA fragment is inserted into?

A bacterial plasmid

Describe the stages involved in inserting a DNA fragment into a bacterial plasmid. (4 marks)

• Same restriction endonuclease that was used to produce the DNA fragment is used to cut out a section of the plasmid

• Sticky ends of DNA fragment and sticky ends of plasmid bind via complementary base pairing

• DNA ligase is used to anneal DNA fragment and plasmid by binding their sugar-phosphate backbones together

• Gene has been incorporated into the plasmid so plasmid is said to be recombinant

What is transformation?

When the recombinant plasmids are reintroduced into bacterial cells

Which type of plasmids are recombinant plasmids typically made from, and why?

• Plasmids containing the gene for antibiotic resistance

• Antibiotics can be used during transformation to see which bacterial cells have taken up the recombinant plasmid

What are the three types of marker genes which may be used to see which bacterial cells have taken up the recombinant plasmid?

• Antibiotic resistance marker genes

• Fluorescent markers

• Enzyme markers

What are bacterial cells placed on during transformation?

A medium containing calcium ions

Why are bacterial cells placed on a medium containing calcium ions during transformation?

• Calcium ions make the bacterial cell membrane more permeable

• This allows plasmids to pass through the cell-surface membrane into the cytoplasm of the bacterial cell