Site author Richard Steane
The BioTopics website gives access to interactive resource material, developed to support the
learning and teaching of Biology at a variety of levels.
Recombinant DNA technology
This process involves the transfer of DNA from one organism to another, usually of a different species. Of course, it is not all the DNA from the source or donor organism that is transferred, but merely fragments of it, such as specific genes.
The recipient organism can be described as transgenic, and it is expected that the DNA that is transferred can potentially be processed in the same way as its normal DNA. This is because the genetic code is universal, and so the base code of the introduced DNA can be transcribed into RNA, which can be translated into amino acids, forming protein specified by the transferred gene. It is said that transcription and translation mechanisms are also universal, even though the enzymes, ribosomes, etc, are produced by the host cell.
In fact genes can be transferred between quite unrelated organisms, for example different species of plants, microorganisms and animals, and this is a basic process in genetic engineering.
It is usually necessary to transfer genes to the cells of animals or plants at an early stage in their development if it is needed to develop the desired characteristics in the adult stage. Introducing genes to specific cells and organs after their differentiation and development is a much more unreliable process.
A number of crop plants have been genetically modified (GM), including plants that are at least partially resistant to attack by insect pests or herbicides and some large companies with different interests, such as chemical companies hve become involved.
Some of these developments have proved to be unacceptable to consumers who prefer more natural products, and others are concerned about the ecological consequences of the use of these products. In fact it is true to say that there are economic, social and ethical arguments for and against GM crops and genetic modification in general .
The term Genetic recombination is used to describe the mutual transfer of genetic material between non-sister homologous chromatids as a result of crossing over in Prophase I of meiosis. This - 'natural' process - establishes a new sequence of alleles.
Minor (?) variations in the genetic code are found in mitochondria and certain ciliate protozoa - mainly differences in start and stop codons, although some blocks of codons code for different amino acids. In bacteria and (mammalian) mitochondria the start codon gives formylmethionine instead of methionine.
Sources of DNA fragments
In the laboratory, DNA may be obtained from a number of sources
mRNA can be collected in a cell which is expressing the gene in question, so it is actively transcribing mRNA ready for use in translation. This can be converted into complementary DNA (cDNA), by using the (viral) enzyme reverse transcriptase.
DNA can be cut into fragments, hopefully containing a gene which is under investigation, using restriction enzymes (restriction endonucleases).
These enzymes cut DNA at specific nucleotide sequences (more below)
A gene machine - a DNA synthesiser - can be used to assemble the DNA from individual nucleotides, under computer control.
In forensic work, small quantities of DNA may be collected and these may need to be amplified by the PCR technique (below), before being used in identification.
In DNA sequencing work, larger amounts of genetic material are collected, and this may also need to be amplified by PCR, and data from this may be combined to give the full sequence.
Sources of mRNA
Within the human body there are a number of different cell types, all containing the same DNA.
But only those cells that need to be producing a specific protein product will be transcribing particular sections of that DNA - genes - to make mRNA, and then translating that to make the specific polypeptide/protein that is characteristic of that gene. This mRNA may be extracted from cells of active tissues and processed to make DNA corresponding to the specific gene under investigation.
Reversing the gene copying process to make DNA
The action of reverse transcriptase enzymes is dealt with in the section on Replication of HIV in helper T cells
on this site.
The H-8 is 'a milestone of closed-column oligo production'.
It looks like 4 nucleotides are drawn in from the bottles at the front, and the product is collected in the middle.
('Oligos' for short) can be synthesised from individual nucleotides.
These short lengths of DNA or RNA can be used as templates, primers and c-DNA.
It is useful to be able to obtain larger amounts of DNA for analysis, or to be able to pass it on for other observations using other organisms.
There are several techniques for this - either in vitro (chemical) or in vivo (biological) in nature.
Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is an in vitro
method to amplify DNA fragments.
This relies on the structure of the DNA molecule, often described as a double helix.
The sample is mixed with enzymes, primers, nucleotides and buffers. A dye that fluoresces when bound to DNA may be added, so as to monitor the progress of the reaction.
When explaining this process, it is important to stress the separation and joining of nucleotides rather than just bases
The procedure has three steps, which can be repeated several times.
- The two strands of the DNA double helix are separated by raising the temperature to about 95 °C for 1-10 minutes.
This causes hydrogen bonds between bases to break, so that single strands of DNA move apart. It does not break the 'backbone' of the molecule, which consists of a chain of deoxyriboses alternating with phosphates, held together by covalent phosphodiester bonds. These are firmly attached to the bases, part of which are exposed in a row. This step is referred to as denaturation or 'DNA melting', but it is not as destructive as the denaturation of protein.
- The temperature is lowered slightly (about 55 °C) for the next stage for 20-40 seconds.
Primers can then migrate into position near the 5' end of the single DNA strands, and bind as a result of complementary base pairing.
This step is referred to as annealing.
DNA polymerase attaches to the single DNA strand, next to the double-stranded section formed by the primer, and as it moves along the strand from 5' to 3' ends,
individual DNA nucleotides (actually triphosphates- dATP, dGTP, dCTP, dTTP) are brought in, joining on to the exposed bases by complementary base pairing and hydrogen bonding.
The formation of phosphodiester bonds between them is catalysed by the enzyme DNA polymerase, and energy is provided as double phosphate units (PPi- pyrophosphate) are released, leaving a single phosphate group on each nucleotide. In this way, the second strand is formed, rebuilding the double helix.
This process is performed at a slightly higher temperature (5-10 min at 68-72 °C.).
It is normally carried out using a heat-stable DNA polymerase.
This step is referred to as elongation. The intensity of fluorescent light emitted can be measured to see what stage the process has reached.
Of course there are now two copies of the original molecule.
So repeating the stages above will result in four copies, then eight, then sixteen - an exponential increase.
In fact there are some refinements to the technique which improve the speed of the process:
How many DNA molecules would theoretically be produced from one molecule of DNA after
- Using DNA polymerase enzyme extracted from a thermotolerant microorganism, which is unaffected by the highest temperature.
This is usually called Taq, as it is isolated from the thermophilic bacterium Thermus aquaticus.
- Addition of single-stranded DNA primers which attach to the end of the exposed DNA single helix, acting as a starting point for the DNA polymerase which moves along the strand
- Thermal cycling provides pulses of heat to separate the strands, and other temperatures for the other steps.
- The solution is buffered to keep pH stable
- It is normal to perform a set number of heating cycles, such as 25 or 35.
? > 225 - which is 33554432 (3.4× 107)
? > 235 - which is 3.4 × 1010
In reality, the process only produces a certain number of DNA molecules, and it reaches a plateau after some time.
What practically reduces the actual number?
nucleotides and/or templates being used up
Some of the principles behind the polymerase chain reaction have been covered in a number of topics on this site (links below
Structure of Nucleic Acids (DNA and RNA)
Reverse transcriptase-polymerase chain reaction
This is a modification of PCR to detect RNA in a tissue sample, in order to identify its source (e.g. Coronavirus or HIV which are both RNA viruses), or to measure accurately the amount present.
This involves the enzyme reverse transcriptase which converts the RNA into DNA which can then be amplified as explained alongside.
However, the first stage is the removal of any DNA in the tissue sample, which would be amplified along with DNA produced from the RNA.
DNA must be hydrolysed by incubating it with the DNase enzyme, then the sample is heated to destroy the enzyme.
Then the sample is incubated with reverse transcriptase in the presence of deoxyribose nucleotides, in order to convert any RNA into DNA.
Next it is given the standard treatment for PCR (using DNA polymerase, deoxyribose nucleotides, and thermal cycling) in order to amplify the DNA which has been reverse-transcribed from the RNA. In identifying viral infections it is normal to use a primer that is specific to the (nucleic acid of) the virus in question
It is normal to add a dye that fluoresces when it binds with DNA, so that the final DNA concentration can be calculated by measuring the intensity of light emitted. The starting RNA concentration can be derived from this, making allowances for the number of heating cycles involved.
Alternatively radioactive probes may be used and the radioactivity used in measuring the quantity of DNA fragments present, possibly in conjunction with electrophoresis to separate the sample into different size fractions.
Use of enzymes to transfer sections of DNA
Enzymes may be used not only to extract DNA fragments, as mentioned above, but also to insert them into vectors, by a 'Cut and Paste' technique.
Cut . .
There are a number of restriction endonuclease enzymes, each of which cuts DNA at a certain base sequence. The majority cut the two DNA strands asymmetrically, leaving protruding single-stranded sections - so-called 'sticky ends'. See EcoR1, opposite
If the base sequence of the gene is known, it is possible to choose a restriction endonuclease that cuts at or near both the start and end of the gene, rather than in the middle of it, and this can be used to cut the gene out of its source.
The same enzyme can be used to cut open the DNA of the target organ or a vector which may be used to transfer the gene into its new cell.
The DNA fragment may become attached to the gap in its intended destination as a result of complementary base pairing between the exposed sticky ends, and weak hydrogen bonds hold them together.
. . and Paste
Another enzyme DNA ligase can be used to complete the joining of the two sections by creating stronger phosphodiester bonds at either end of the insertion.
The 'cut-and-paste' process of inserting genes may not reliably affect all the cells expected.
It is normal practice to include marker genes in order to detect cells or organisms that have become genetically modified.
These may be antibiotic resistance genes in bacterial plasmids, or protein pigments.
Bacteria that have taken up a plasmid containing an antibiotic resistance gene will not be killed when transferred to a culture medium containing that antibiotic, so they are more likely to have the required DNA in their cells.
Alternatively, if the gene is inserted into the middle of a subsidiary gene, this can be predicted by loss of this genes activity.
Why is it important to use the same restriction enzyme to cut the gene and the target DNA?
> To ensure that sticky ends are complementary, so the join will be exact
More about Restriction Enzymes (restriction endonucleases)
These are produced by different bacteria, and it is thought that they act against viruses which might infect them. In fact the term comes from the observation that some bacterial strains, when infected with bacteriophage viruses, did not produce as many virus particles as others, so the becteria were restricting the production of viruses.
Thousands of different restriction endonuclease enzymes are known, and hundreds of them have become commercially available tools used by scientists involved in genetic engineering.
These enzymes attach to DNA and respond to particular base sequences within it. Each one has a different recognition site and it breaks the DNA at a specific cleavage site, usually nearby. They mostly require Mg2+ as a cofactor. In fact this ion brings in water molecules which are required to hydrolyse phosphodiester bonds at the cleavage site.
Restriction enzymes may be classified into different groups, depending on composition, and characteristics of the cleavage site. They may also require different cofactors, such as ATP.
The main group of interest, type II, cleave within or at short specific distances from the recognition site, and do not require ATP.
Some restriction enzymes cut the two strands of DNA at different points so there is an overhang of single stranded DNA at each end of the cut, resulting in 'sticky ends'.
This is because the DNA is cut within a short so-called palindromic sequence, in which the base sequence on one strand is repeated (in the opposite direction) on the other strand. Recognition seqences of 6 or 4 bases are common.
Some leave this overhang on the 5' end, whereas others leave the overhang on the 3' end.
Others cut evenly across, leaving 'blunt ends'.
Cleavage of the DNA results in fragments with phosphate groups at their 5' end, and hydroxyl groups at their 3' end.
Restriction enzymes are named according to the bacterial strains from which they originate.
EcoR1 comes from Escherichia coli
EcoR1 identifies the palindromic sequence GAATTC in DNA. The enzyme is a dimer - there are two identical sections, each with their own active site.
One of these cuts the DNA backbone between the G and A, so one section of DNA ends up with a protruding single strand section of AATT.
The other strand of DNA will have the sequence CTTAAG and again the (other part of the) enzyme cuts between A and G, so a section of DNA with a protruding single strand section of TTAA is produced.
This is summarised below using the 5' and 3' notation to show the direction of the DNA strands.
Obviously the deoxyribose and phosphate groups are omitted, as well as the DNA nucleotides on either side of the recognition sequence.
What do the vertical lines | signify ? > (enzymic breaking of stronger) phosphate bonds
What do the horizontal lines signify ? > (separation of much weaker) hydrogen bonds
How do we describe the direction of the overhanging sticky ends above? > -5'
Any DNA with an exposed single strand section of AATT could bind with another section of DNA which has an exposed single strand section of TTAA, before forming a strong covalent linkage.
Explain this possible initial pairing with reference to formation of bonds.
> This is 'complementary base pairing' (A with T in this case)
> This section is held together by hydrogen bonds NOT PHOSPHODIESTER BONDS
EcoRV also comes from Escherichia coli, and the V is read as 'five'.
Its recognition sequence is 5'-GAT|ATC-3' and it cuts both strands at the same place, leaving 'blunt ends'.
HindIII comes from Haemophilus influenzae d
This has the recognition sequence 5'-AAGCTT-3', and it cuts after the first A:
5'-A|A G C T T-3'
3'-T T C G A|A-5'
BamH1 from Bacillus amyloliquefaciens has the recognition sequence 5'-GGATCC-3', and it cuts after the first G:
5'-G|G A T C C-3'
3'-C C T A G|G-5'
PstI comes from Providencia stuartii.
Its recognition sequence is 5'-CTGCAG-3' and it cuts betweeen A and G, leaving an overhang on the 3' end.
5'-C T G C A|G-3'
3'-G|A C G T A-5'
More about DNA ligase
DNA ligase enzymes repair DNA by joining 'nicks' in the deoxyribose-phosphate backbone. There are several types dealing with single-strand breaks in DNA, catalysing a reaction between a 3'-OH group on one deoxyribose and a 5'-monophosphate on another deoxyribose.
They form ester bonds between the phosphate group already on one nucleotide and the exposed 3'-hydroxyl group on the next deoxyribose section, using ATP as cofactor.
In cell division, DNA ligase is involved in filling in gaps between Okazaki fragments on the 'lag' strands of replicating DNA.
If a section of DNA (corresponding to a gene of interest to scientists or physicians) can be inserted into the cells of an easily cultured organism such as a bacterium, then the gene may be effectively amplified as the transformed cells reproduce themselves. This is sometimes described as gene cloning. These techniques involve the use of vectors to introduce the extra DNA into the cells.
Promoter and terminator regions may be joined to either end of the selected fragments of DNA.
Some bacteria have extra DNA loops in addition to their main DNA which codes for proteins they use in their normal functioning.
These loops or plasmids are sometimes associated with extra characteristics such as antibiotic resistance, and they can be passed from cell to cell, and even between different species of bacteria.
In the laboratory, plasmids can be extracted from bacterial cells following treatment with heat or calcium ions (Ca2+), and then cut with enzymes so as to allow extra DNA to be inserted. The plasmids may be taken up by some, but not all, of the bacteria in another culture. Sometimes bacterial cell walls are also weakened by treatment with heat or calcium ions, or by electric current (electroporation) to allow the entry of plasmids.
When recombinant DNA is introduced into bacteria, some bacteria are successfully transformed as this DNA has been taken up by plasmids, but some bacterial cells remain non-transformed.
The use of bacteria containing antibiotic resistance genes is controversial in some contexts, but it is useful in the laboratory.
What are negative issues associated with these genes?
> Antibiotic resistance genes may be passed to bacteria in the environment
> These may cause infections which cannot be controlled by this antibiotic
What is the advantage to using them in recombinant DNA technology?
> Growing bacteria in medium containing the antibiotic will select for cells which have the plasmid, and may have taken up the required gene.
Others - without the plasmids - will be killed by the antiobiotic.
> This selects for/identifies genetically modified (GM) cells
The gall-forming bacterium Agrobacterium tumefaciens has Ti (tumour inducing) plasmids that can be used to transfer genetic material into plant cells.
Viruses insert their DNA into cells they infect. Bacteriophages are viruses that infect bacteria, and sometimes they insert their nucleic acid into the DNA of the bacteria, and this extra material will be replicated along with the normal bacterial DNA as cells reproduce.
Copies of DNA may be inserted into nuclei of body cells from domesticated animals, and these nuclei can be inserted into egg cells from which the nuclei have been removed. The egg cells divide to form embryos which can be implanted into the uterus of a surrogate animal.
Artificial chromosomes consisting of sections of DNA may be used to incorporate DNA fragments into target cells.
The pBR322 plasmid is widely used as a cloning vector
The genes amp and tet give resistance to the antibiotics ampicillin and tetracycline, and ori stands for origin of replication.
Target sites for a number of restriction enzymes are shown in blue.
Genetic engineering applied to blood clotting proteins
This has been performed experimentally using sheep and pigs in an attempt to produce milk containing useful proteins by targetting mammary gland tissue.
The genes responsible for one of the blood clotting proteins factor VIII and factor IX, absent in haemophilia, have been combined with promoters taken from the mammary gland cells which normally start transcription of the genes which code for milk proteins. In some cases a gene taken from a jellyfish was attached to the human Factor IX gene. This gene codes for a protein that glows green under fluorescent light.
What are the advantages of producing these proteins in milk?
> They can be produced over an extended length of time ('lactation')
> Protein is easily extracted (by milking the animal) without needing to remove and break open cells
> It does not involve slaughter of animals concerned
What is the function of the promoter in this combination?
> To turn on production of the required protein in mammary tissue ONLY
What is the advantage to the attachment of the jellyfish gene?
> It acts as a marker
> To show that the (human) factor IX gene has been taken up/ and expressed
> It is only worth implanting embryos that show fluorescence because they contain the jellyfish gene (as well as the required factor IX gene ?)
The first approved genetically modified food product
Trying to make sense of these strands - but your RNA will end up in a tangle
The coding strand shown in blue at the bottom normally transcribes mRNA, but the other strand also produces RNA, because a promoter has been spliced in.
The two strands of RNA combine to form duplex (double-stranded) RNA, which cannot be translated into the normal enzyme.
was a variety of tomato
that was genetically engineered to produce less of the enzyme polygalacturonase which plays a part in fruit softening.
So-called antisense RNA
was produced which reduced this enzyme activity by 70-90%.
See the diagram alongside
Why does the duplex RNA form?
> Each RNA strand has a complementary base sequence to the other
> And hydrogen bonds form between the bases along the strands
How does RNA being duplex interfere with translation?
> RibosOMe will not fit on double-stranded RNA
> There are no exposed bases
> Exposed mRNA bases are needed for tRNA to pair with, so no amino acids are brought in to form a polypeptide
Why is a promoter inserted into the top DNA strand?
> to give a signal for the gene to be transcribed
What is the significance of the promoter being at the 'wrong end of the DNA sequence' at the top ?
> RNA polymerase transcribes RNA from DNA in only one direction - from 3' to 5' on the DNA
> And the DNA of the normally non-coding strand runs in the opposite direction to the coding strand - anti-parallel
Other related topics on this site
(also accessible from the drop-down menu above)
Using genome projects
Interactive 3-D molecular graphic models on this site
(also accessible from the drop-down menu above)
The DNA molecule - rotatable in 3 dimensions
Role of Recombinant DNA Technology to Improve Life
Making recombinant DNA
New genetic codes in mitochondria and ciliate protozoa
Reduction of polygalacturonase activity in tomato fruit by antisense RNA
Thomas Brock, Whose Discovery Paved the Way for PCR Tests, Dies at 94 - News item about the microbiologist who found heat-resistant bacteria in a hot spring at Yellowstone National Park