A-level Biology - 3.4.10 Recombinant DNA Technology

DNA from 2 different sources are combined

Transferring a fragment of DNA from one organism to another

∵ genetic code is universal

(same DNA base triplets code for same amino acids in all living things)

∵ transcription and translation mechanisms are pretty similar

Using Reverse Transcriptase

Using Restriction Endonuclease Enzymes

Using a ‘Gene Machine’

Many mRNA molecules (complementary to a gene)

Makes DNA from an RNA template

complementary DNA (cDNA)

mRNA is isolated from cells

Its mixed with free DNA nucleotides and reverse transcriptase

Reverse transcriptase uses mRNA as a template to synthesis a new strand of cDNA

DNA polymerase is used to build up the complementary base pairings with cDNA = DNA is formed

palindromic

Sequences that consist of antiparallel base pairs

(Base pairs that read the same in opposite directions)

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) DNA at these places

∵ shape of recognition sequence is complementary to enzyme’s active site

If recognition sequences are present either side of DNA fragment

DNA sample is incubated with specific restriction endonuclease & it cuts DNA fragment out via hydrolysis reaction

Sometimes cut leaves sticky ends - small tails of unpaired bases at each end of the fragment

Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences

Technology that enables fragments of DNA that can be synthesised from scratch without need for pre-existing DNA template

Database contains necessary info to produce DNA fragments

Can produce DNA sequences that don’t exist naturally

Sequence required is designed

1st nucleotide in sequence is fixed to some sort of support

e.g. a bead

Nucleotides are added step by step in correct order, in a cycle of processes that include adding protecting groups

Short sections of DNA (called oligonucleotides - 20 nucleotides long) are produced

Once complete, they’re broken off from support and all protecting groups are removed

Oligonucleotides can then be joined together to make longer DNA fragments

Protecting groups make sure nucleotides are joined at the right points, to prevent unwanted branching

In Vivo Amplification

In Vitro Amplification

DNA Fragment is Inserted into Vector

Vector Transfers DNA Fragment into Host Cells

Identifying Transformed Host Cells

Can be plasmids or bacteriophages (viruses that infect bacteria)

When a gene is added from another organism to the plasmid

Vector DNA is cut open using same restriction endonuclease that was used to isolate DNA fragments containing the target gene

So sticky ends of vector are complementary to sticky ends of DNA fragment containing the gene

Vector DNA and DNA fragment are mixed together with DNA ligase

DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA

aka ligation

Host cells are to be persuaded to take in the plasmid vector and its DNA

Bacteriophage infect host bacterium by injecting its DNA into it

Phage DNA (with the target gene in it) then integrates into bacterial DNA

transformed

Marker genes

Inserted at same time as target gene (i.e. gene to be cloned)

∴ transformed host cells will contain target gene and marker gene

Agar plates

Each cell divides and replicates its DNA, creating colony of cloned cells

Genes that code for antibiotic resistance

Genes that code for fluorescence

Enzyme markers

Host cells are grown on agar plates containing specific antibiotic so only transformed cells with marker genes will grow

Under UV light only transformed cells will fluoresce

Gene produces lactase and lactase will turn colourless substate blue

Bacteria are placed into a medium containing e.g. ampicillin, with all bacteria that have taken up the plasmid (with gene for resistance to ampicillin) surviving

Each separate cell then grows into a genetically identical colony

A tiny sample of each colony is then placed into a medium containing tetracycline

The cells that are destroyed contain the recombinant DNA

Shows new gene has been taken up as gene for resistance to tetracycline is made useless

Vector contains specific promoter and terminator regions

DNA fragment won’t be transcribed by host cell and protein won’t be made

DNA sequences that tell enzyme RNA polymerase when to start producing mRNA

(Terminator regions tell it when to stop)

Promoter and terminator regions may be present in the vector DNA or may be added in along with the fragment

Use polymerase chain reaction (PCR)

(copies of DNA fragments are made outside of living organisms)

It can be used to make millions of copies of a fragment of DNA in a few hours

Reaction mixture set up containing DNA sample, free nucleotides, primers and DNA polymerase

DNA polymerase creates new DNA strands

DNA mixture is heated to 95°C to break hydrogen bonds between 2 strands of DNA

Strands separate

Mixture is cooled to between 50°C and 65°C so primer can bind (anneal) to strands

Nucleotides attach by complementary base pairing

Reaction mixture is heated to 72°C so DNA polymerase can work

DNA polymerase join free DNA nucleotides alongside each template strand 2 new copies of fragment of DNA formed & 1 cycle of PCR is complete

Cycle starts again, with mixture heated to 95°C and all 4 strands (2 original and 2 new) are used as templates

Each PCR cycle doubles the amount of DNA

16 DNA fragments

1st cycle = 2 x 2 = 4 DNA fragments

2nd cycle = 4 x 2 = 8 DNA fragments

3rd cycle = 8 x 2 = 16 DNA fragments

Through genetic engineering

Can be made using same technology as in vivo cloning

e.g. foreign DNA inserted into microorganisms to produce lots of useful protein

Gene codes for desirable protein inserted into plasmid

Plasmid is added to bacterium and bacterium is used as vector to get gene into plant cells

If right promoter region has been added along with gene, transformed cells will be able to produce the desired protein

Gene codes for desirable protein can be inserted into early animal embryo or into egg cells of a female

If gene inserted into a very early embryo, all body cells of resulting transformed animal will contain the gene

Inserting into egg cells = when female reproduces = all cells of offspring contain the gene

exactly which of animal's body cells the protein is produced in

It can be harvested more easily

Damages the organism

Agriculture crops can be transformed to they give higher yields or are more nutritious

Means plants can be used to reduce risk of famine and malnutrition

Crops can be transformed to have pest resistance = fewer pesticides are needed

Reduces costs and reduces any environmental problems associated with using pesticides

Industrial processes often use enzymes

Enzymes can be produced from transformed organisms = produced in large quantities for less money = reducing costs

Many drugs and vaccines are produced by transformed organisms, using recombinant DNA technology

Can made quickly, cheaply and in large quantities using this method

So more people can afford them

Recombinant DNA technology has the potential to be used in gene therapy to treat human diseases

Altering the defective genes (mutated alleles) inside cells to treat genetic disorders and cancer

Farmers might plant only 1 type of transformed crop (monoculture)

Makes whole crop vulnerable to same disease ∵ plants are genetically identical

Monoculture also reduces biodiversity = damages the environment

Anti-globalisation activists oppose globalisation

A few, large biotechnology companies control some forms of genetic engineering

As use of this technology increases = companies get bigger and more powerful

May force smaller companies out of business

Without proper labelling, some people think they won't have a choice about whether to consume food made using genetically engineered organisms

Some consumer markets (e.g EU) won't import GM food and products

Can cause economic loss to producers who have traditionally sold to those markets

Companies who own genetic engineering technologies may limit the use of technologies that could be saving lives

Some people worry this technology could be use unethically

e.g. make designer babies = illegal

Enables replication/sequencing to start

Short lengths of single-stranded DNA that are complementary to a particular sequence of DNA