Recombinant DNA Technology and Gene Therapy

What does recombinant DNA technology involve?

Transfer of DNA fragments from one organism/ species to another, involving a number of processes. Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient organism

What are the 5 processes involved?

• Isolation/production of DNA fragments that have the gene for the desired protein

• Insertion if the DNA fragments into a vector

• Transfer of DNA into suitable host cell

• Use of marker genes to detect genetically modified cells or organisms

• Growth/ cloning of population of host cells

What are the 3 ways to isolate DNA fragments?

• Using reverse transcriptase

• Using restriction endonucleases

• Using the gene machine

What is the process of using reverse transcriptase?

• Isolate mRNA from a cell that readily synthesis the protein coded for by the desired gene (mRNA easily extracted as these cells have large quantities)

• Mix mRNA with DNA nucleotides and reverse transcriptase (the reverse transcriptase uses mRNA as a template to synthesis a single strand of complementary DNA)

• DNA polymerase is used to build up the complementary nucleotides on the cDNA template, forming a second strand of DNA. This double strand of DNA is the required gene

What is the process of using restriction endonuclease?

• Restriction endonuclease enzymes cut DNA at the specific base ‘recognition sequence' either side of the desired gene. The shape of the recognition side is complementary to the active site of the enzyme

• Many enzymes cut in a staggered fashion, forming complementary sticky ends

Why are sticky ends called sticky ends?

Their exposed bases readily form hydrogen bonds with the complementary bases in the sticky ends of other molecules, cut by the same restriction endonuclease

How can fragments of DNA be extracted using a gene machine?

• Synthesises fragments of DNA quickly and accurately from scratch, without needing a DNA template (amino acid sequence of the protein is determined, allowing the base sequence to be established)

• These do not contain introns so scan be transcribed and translated by prokaryotes

• This is a slow and expensive process, so isn’t used

What are the advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells?

• Much more mRNA than DNA in cells making the protein than DNA, so more easily extracted

• In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns, so can be transcribed and translated by prokaryotes who cant remove introns by splicing

Name an in vitro and in vivo technique used to amplify DNA fragments

In vitro (outside living organism)- polymerase chain reaction

In vivo (inside a living organism)- culturing transformed host cells e.g. bacteria

How can fragments of DNA be amplified in vivo?

Adding of promoter and terminator regions

• this happens at the end of isolating your DNA fragment

• once you have obtained your required DNA fragment, we need to attach a promoter region and terminator region

• The promoter region is added so the DNA fragment can be transcribed onto an mRNA strand in order to make the protein, so it starts the process, by allowing RNA polymerase to bind to DNA. Can be selected to ensure gene expression happens only in one specific cell type

• The terminator region is added to stop transcription at the appropriate point, by stopping RNA polymerase

Summarise the steps involved in amplifying DNA fragments in vivo

• Add promoter and terminator regions to DNA fragments

• Insert DNA fragments and marker genes into vectors using restriction enzymes and ligases

• Transform host cells (bacteria) by inserting these into vectors

• Detect genetically modified cells by identifying those which contain the marker gene

• Culture these transformed host cells, allowing them to divide and form clones

Following this, DNA can be extracted from the host cells if needed or host cells can produce a protein coded for by a gene in DNA fragment

What is the role of vectors in recombinant DNA technology?

To transfer/ transport DNA directly into host cells e.g. normally plasmids or viruses

Explain how DNA fragments can be amplified in vitro (PCR)

Mixture heated to 95 degrees

• This separates the DNA strands

• And breaks the hydrogen bonds between complementary bases

Mixture cooled to 55 degrees

• This allowed primers to bind to DNA fragment template separated strand

• By forming hydrogen bonds between the complementary bases

Mixture heated to 72 degrees

• Nucleotides align next to complementary exposed pairs

• DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

This cycle is repeated, in every cycle the amount of DNA doubles, causing an exponential increase (2ⁿ)

Explain the role of primers in PCR

• Primers are short, single-stranded DNA fragments

• They are complementary to DNA base sequence at edges of region to be copied/ start of desired gene

• Allowing DNA polymerase to bind to start synthesis, as before it could only add nucleotides onto preexisting triple codon

• There are two different primers: forwards and reverse, which are required as base sequences at each end of DNA strand are different

Explain why transferred DNA can be translated within cells of recipient organisms

• Genetic code is universal

• Transcription and translation are universal

Explain the role of enzymes in inserting DNA fragments into vectors

A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have the same recognition site for the same restriction endonuclease

• Restriction endonuclease cut out the gene from vector DNA/ plasmid; the same enzyme is used to cut the gene out of foreign DNA so vector DNA and new DNA fragment have sticky ends that can join by complementary base pairing with hydrogen bonds

• DNA ligase joins the DNA fragment to the vector DNA, forming phosphodiester bonds between adjacent nucleotides

Suggest one reason why DNA replication eventually stops in PCR

There are a limited numbers of primers and nucleotides, which are eventually all used up

Describe how host cells are transformed using vectors

• Plasmids enter cells (e.g. following heat shock in a calcium ion solution)

• Viruses inject their DNA into cells which is then integrated into host DNA

Explain why marker genes are inserted into vectors

• To allow detection the cells which have been genetically modified

Examples

• If marker gene codes for antibiotic resistance, cells that survive antibiotic resistance= modified

• If marker gene codes for fluorescent proteins, cells that fluoresce under UV light= modified

• As not all cells/ organisms will take up the vector and be transformed

How can recombinant DNA technology be useful in medicine?

• Genetically modified (GM) bacteria produce human proteins

  • More ethically acceptable than using animal proteins and less likely to cause allergic reactions

• GM animals/ plants produce pharmaceuticals

  • Cheaper

• Gene therapy

How can recombinant DNA technology be useful in agriculture?

• GM crops resistant to herbicides

  • Only weeds will be killed when crops sprayed with herbicide

• GM crops resistant to insect attack

  • Reduces use of insecticide

• GM crops with added nutritional value

• GM animals with increased growth hormone production e.g. salmon

How can recombinant DNA technology be useful in industry?

GM bacteria produce enzymes, which are used in industrial processes and food production

Describe gene therapy

• Introduction of new DNA into cells, often containing healthy/ functioning alleles

• To overcome effect of faulty/ non-functional alleles in people with genetic disorders e.g. cystic fibrosis

(If body cells are altered/ have been through gene therapy, the changes are NOT heritable. Gene therapy is currently illegal on gametes).

Suggest some issues associated with gene therapy

• Effect is short-lived as modified cells have a limited lifespan, so required regular treatment e.g. T cells

• Immune response against genetically modified cells or viruses due to recognition of antigens

• Long term effect/ side effects are unknown

  • DNA may be inserted into other genes and disrupt them, interfering with gene expression

Why may humanitarians support recombinant DNA technology?

• GM crops increase yield of crops

  • Could in turn increase global food production

  • Reduced risk of famine/ malnutrition

• Gene therapy has potential to cure some genetic disorders

• Pharming makes medicine available to more people as it becomes cheaper

Why may environmentalists and anti-globalisation activists oppose recombinant DNA technology?

• Recombinant DNA may be transferred to other plants

• Potential effects on food webs and reduce biodiversity

• Large biotech companies may control the technology and own patents