NEW Recombinant DNA Technology

8.
4 Gene technologies
8.4.1 Recombinant DNA technology
Recombinant DNA technology
● Transfer of DNA fragments from one organism or species, to another

● Transferred DNA can be translated within cells of recipient (transgenic) organism because:
1. Genetic code is universal
2. Transcription and translation mechanisms are universal
Methods of producing DNA fragments
Method 1 - Using restriction enzymes to cut a

fragment containing desired gene from DNA
● Different restriction enzymes cut DNA at

specific base ‘recognition sequences’
○ Shape of recognition site
complementary to active site
● Many cut in a staggered fashion
● Forming ‘sticky ends’ (single stranded
overhang)
Method 2 - Conversion of mRNA to complementary

DNA (cDNA), using reverse transcriptase
1. mRNA isolated from a cell that readily synthesises protein coded for by desired gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses mRNA
as a template to synthesise a single strand of cDNA
3. DNA polymerase forms second strand of DNA using cDNA as a template
Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut gene from DNA
● More mRNA in cell than DNA → easily extracted

● In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns
○ So can be transcribed/translated by a prokaryote who can’t remove introns by splicing
Method 3 - Creating the gene in a ‘gene machine’
● Synthesises fragments of DNA from scratch without need for a pre-existing DNA template
○ Amino acid sequence of protein determined allowing nucleotide sequence to established
● DNA fragments produced quickly / accurately
● No introns → can be transcribed/translated by a prokaryote who can’t remove introns
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The principles of the polymerase chain reaction (PCR) as an in vitro method to
amplify DNA fragments
PCR: amplification of specific DNA fragments
Reaction mixture: DNA fragment + DNA polymerase (taq polymerase)

+ (forward/reverse) primers + nucleotides
1 Strand separation ● Separates DNA strands

o
95 C ● By breaking hydrogen bonds between bases
2 Primer binding ● Allows primers to bind to DNA fragment template strand

o
55 C ● By forming hydrogen bonds between complementary bases
3 Synthesis of DNA ● Optimum temperature for DNA polymerase (taq polymerase)

o
72 C ● Nucleotides align next to complementary exposed bases
● DNA polymerase joins adjacent nucleotides forming phosphodiester bonds
● Cycle repeated 30-40 times to make many copies of DNA fragment

● In every cycle, amount of DNA doubles so number of molecules made = 2n, where n = number of cycles
○ Causing an exponential increase
● Stops when run out of nucleotides / primers
Roles of primers
● Primer = short, single stranded DNA fragment

● Complementary to template DNA at edges of region to be copied / start of desired gene
● Allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)
● Two different primers required (forward and reverse)
○ Because DNA strands run in anti-parallel, but DNA polymerase can only run in one direction
Culture of transformed host cells as an in vivo method to amplify DNA fragments
Step 1 - promoter and terminator regions added to DNA fragment
Promoter regions ● DNA sequences that tell RNA polymerase when to start transcription to produce
mRNA
● Can be only activated in specific cell types → control which of body cells protein
produced in
Terminator regions ● Tell RNA polymerase when to stop
Step 2 - restriction endonucleases and ligases used to insert DNA fragment into vectors
A. Restriction endonucleases
● Vector DNA and DNA fragment cut using

same restriction enzyme
● Vector DNA and DNA fragment have
complementary ‘sticky ends’ (single
stranded ends with bases exposed) →
complementary base pair
B. Role of DNA ligases
● DNA ligase joins DNA fragment to vector

DNA
● Forms phosphodiester bond between
adjacent nucleotides on sticky ends
(condensation reaction)
Step 3 - Host cells transformed using vectors
Vector: carrier of / transports DNA into host cell

eg.plasmids or virus/bacteriophage
If plasmid If virus
● Host cells persuaded to take in plasmid vector ● Bacteriophage vector – infects host bacterium
● Eg. place in ice-cold calcium chloride solution by injecting its DNA into it, then the phage DNA
to make cell walls more permeable, then with target gene integrates into bacterial DNA
plasmids added, and mixture is heat shocked
Step 4 - Marker genes used to detect genetically modified (GM) cells or organisms
Marker genes, inserted into vectors at same time as target gene, are added in order to identify which cells
have the desired gene - not all cells / organisms will take up the vector and be transformed
Gene markers can code for proteins which give:

a. Resistance to an antibiotic (cells surviving exposure are transformed)
b. Fluorescence (fluoresce under UV = transformed)
‘Students should be able to’ - evaluating uses of recombinant DNA technology
Students should be able to:
● Interpret information relating to the use of recombinant DNA technology

● Evaluate the ethical, financial and social issues associated with the use and ownership
of recombinant DNA technology in agriculture, in industry and in medicine
● Balance the humanitarian aspects of recombinant DNA technology with the opposition
from environmentalists and anti-globalisation activists
● Relate recombinant DNA technology to gene therapy
Examples of applications in agriculture
● GM crops resistant to herbicides → can spray crop with herbicide and only weeds are killed
● GM crops resistant to insect attack → reduced use of insecticide
● GM crops with added nutritional value eg. Golden rice has a precursor of vitamin A
● GM animals with increased growth hormone production eg. Salmon
Examples of applications in medicine
● GM bacteria producing recombinant human proteins eg. insulin to treat type 1 diabetes → less likely
than insulin from pig pancreas to cause allergic reactions and more ethically acceptable
● GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper
● Gene therapy - introduction of new DNA into cells, often containing healthy / functional alleles to
overcome effect of faulty / non-functional alleles eg. in cystic fibrosis / SCID
General issues with gene therapy (depends on context)
● Effect is short lived → requires regular treatment

○ Because cells modified eg. T cells have a limited lifespan
● Immune response against genetically modified cells or viruses
○ As foreign antigen recognised
● Long term effect not known - side effects eg. could cause cancer
○ DNA may be inserted into other genes, disrupting them → interfering with gene expression
Humanitarian aspects - examples of benefits to human welfare (depends on context)
● GM crops increase yields → increased global food production → reduced risk of famine / malnutrition
● Gene therapy has potential to cure many genetic disorders
● ‘Pharming’ makes medicines available to more people as medicines cheaper
Opposition from environmentalists
● Recombinant DNA may be transferred to other plants if interbreed →potentially creating herbicide
resistant ‘superweeds’
● Potential effects on food webs eg. affect wild insects → reduce biodiversity

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8.

Recombinant DNA technology

● Transfer of DNA fragments from one organism or species, to another

Methods of producing DNA fragments

Method 1 - Using restriction enzymes to cut a

● Different restriction enzymes cut DNA at

Method 2 - Conversion of mRNA to complementary

● More mRNA in cell than DNA → easily extracted

Method 3 - Creating the gene in a ‘gene machine’

PCR: amplification of specific DNA fragments

Reaction mixture: DNA fragment + DNA polymerase (taq polymerase)

1 Strand separation ● Separates DNA strands

2 Primer binding ● Allows primers to bind to DNA fragment template strand

3 Synthesis of DNA ● Optimum temperature for DNA polymerase (taq polymerase)

● Cycle repeated 30-40 times to make many copies of DNA fragment

● Primer = short, single stranded DNA fragment

Step 1 - promoter and terminator regions added to DNA fragment

Terminator regions ● Tell RNA polymerase when to stop

● Vector DNA and DNA fragment cut using

B. Role of DNA ligases

● DNA ligase joins DNA fragment to vector

Step 3 - Host cells transformed using vectors

Vector: carrier of / transports DNA into host cell

Gene markers can code for proteins which give:

Students should be able to:

● Interpret information relating to the use of recombinant DNA technology

Examples of applications in agriculture

Examples of applications in medicine

General issues with gene therapy (depends on context)

● Effect is short lived → requires regular treatment

Humanitarian aspects - examples of benefits to human welfare (depends on context)

Opposition from environmentalists

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