Recombinant DNA technology year 2 Flashcards
recombinant DNA
-The DNA of two different organisms that has been combined by isolating genes, cloning them and then transferring them into microorganisms. These microorganisms are then grown to provide a ‘factory’ for the continuous production of the desired protein.
The resulting organism is known as a transgenic or genetically modified organism (GMO)
-All relies on the fact that the genetic code is universal. The making of proteins is universal in that the mechanisms of transcription and translation are essentially the same in all living organisms. As a result, transferred DNA can be transcribed and translated within the cells of the recipient (transgenic) organism and the proteins it codes for can be manufactured in the same way as they would be within the donor organism. This is all indirect evidence for evolution
making a protein using the DNA technology of gene transfer and cloning
- Isolation. of the DNA fragments that have the gene for the desired protein
- Insertion. of the DNA fragment into a vector
- Transformation. The transfer of DNA into suitable host cells
4.Identification. of the host cells that have successfully taken up the gene by the use of gene markers - growth/cloning. of the population of host cells
stage one of producing GM protein
Identification and isolation of DNA fragment containing the gene
Several methods of producing DNA fragments:
* conversion of mRNA to cDNA using reverse transcriptase. Known as cDNA as made up of nucleotides that are complementary to the mRNA
* using restriction endonucleases to cut fragments containing the desired gene from DNA
* creating the gene in a gene machine, usually based on a known protein structure
using reverse transcriptase to form DNA fragments
-coded genetic information of retroviruses is in the form of RNA. However, in a host cell they are able to synthesis DNA from their RNA using reverse transcriptase. It catalyses the production of DNA from RNA
- the mRNA acts as a template on which a single-stranded complementery copy of DNA (cDNA) is formed using reverse transcriptase
- single standed (cDNA) is isolated by hydrolysis of the mRNA with an enzyme
- double stranded DNA is formed on the template of the cDNA using DNA polymerase
a cell that readily produces the protein is selected. These cells have large quantities of the relevant mRNA, which is therfore more easily extracted.
using restriction endonucleases to form DNA fragments
- Bacteria are frequently infected by viruses that inject their DNA into them in order to take over the cell. Some bacteria defend themselves by producing restriction endonucleases to cut up the viral DNA.
-There are many types of restriction endonucleases. Each one cuts a DNA double stand at a specific sequence of bases called a recognition sequence. Sometimes this cut occurs between two opposite base pairs. This leaves two straight edges known as blunt ends
-Other restriction endonucleases cut DNA in a staggered fashion. This leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases
the gene machine
- the desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce. The amino acid sequence of this protein is determined. From this, the mRNA codons are looked up and the complementary DNA triplets are worked out
- The desired sequence of nucleotide bases for the gene if fed into a computer
- the sequence is checked for biosaftey and biosecurity to ensure it meets international standards as well as various ethical requirement
- the computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides which can be assembled into the desired gene
- In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence
- The oligonucleotides are then joined together to make a gene. This gene doesnt have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction
- The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double stranded gene. It then multiples this gene many times to give numerous copies
- Using sticky ends the gene can then be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organism in the future
- The genes are checked using standard sequencing techniques and those with errors are rejected
The advantages of this process are that any sequence of nucleotides can be produced, very short time and with great accuracy. Free of introns so can be transcribed and translated by prokaryotic cells
Cloning
once the fragment with the gene has been obtained, the next stage is to clone so there is a sufficient quantity for medical or commercial use. This can be achieved in two ways:
- in vivo, by transferring the fragments to a host cell using a vector- called in vivo as cloned IN the living organism
- in vitro, using the polymerase chain reaction
Whole point is to amplify the DNA fragments that have just been created through the gene machine, using restriction endonuclease or using reverse transcriptase
importance of sticky ends
- The sequences of DNA that are cut by restriction endonucleases are called recognition sites. If the recognition site is cut in a staggered fashion, the cut ends of the DNA double strand are left with a single strand which is a few nucleotide bases long.
- The nucleotides on the single strand at one side of the cut are obviously complementary to those on the other side as they were previously paired together
- If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complementary to one another. This means that the single-stranded end of any one fragment can be joined (stuck) to the single-stranded end of any other fragment. Once the complementary bases of two sticky ends have paired up, an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the two sections of DNA and so unite them as one
- Sticky ends have considerable importance because provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism
preparing the DNA fragment for insertion
- preparation of the DNA fragment involves the addition of extra lengths of DNA. For the transcription of any gene to take place, the enzyme that synthesis mRNA (RNA polymerase) must attach to the DNA near a gene. The binding site of the RNA polymerase is a region of DNA, known as a promoter. The nucleotide bases of the promoter attach both RNA polymerase and transcription factors and so begin the process of transcription. If we want our DNA fragment to transcribe mRNA in order to make a protein, it is essential that we attach to it the necessary promoter region to start the process
- In the same way as a region of DNA binds RNA polymerase and begins transcription of a gene, another region releases RNA polymerase and ends transcription. This region of DNA is called a terminator. Again we need to add a terminator region to the other end of our DNA fragment to stop transcription at the appropriate point
YOU ARE ADDING PROMOTER AND TERMINATOR REGIONS TO THE DNA FRAGMENTS CREATED
Insertion of DNA fragment into a vector
- Once an appropriate fragment of DNA has been cut from the rest of the DNA and the promoter and terminator regions have been added, the next task is to join it into a carrying unit, known as a vector
- This vector is used to transport the DNA into the host cell. There are different types of vector but the most commonly used is the plasmid. Plasmids are circular lengths of DNA found in bacteria. They almost always contain genes for antibiotic resistance and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop (the enzyme cuts open the plasmid)
- The restriction endonuclease used is the same as the one that cut out the DNA fragment. This ensures that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment. When the DNA fragments are mixed with the opened-up plasmids, they become incorporated into them. Where they are incorporated, the join is made permanent using the enzyme DNA ligase, which catalyses condensation reactions between the DNA, forming phosphodiester bonds between the nucleotides. These plasmids now have recombinant DNA
Introduction of DNA within plasmids into host cells
Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells. This process is called transformation and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions. The calcium ions, and changes in temperature make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm.
Identification of which bacterial cells have taken up the plasmids
However not all the bacterial cells will possess the DNA fragments with the desired gene for the desired protein. Some reasons for this are:
* Only a few bacterial cells take up the plasmids when the two are mixed together
* Some plasmids will have closed up again without incorporating the DNA fragment (close before the DNA enters)
* Sometimes the DNA fragment ends join together to form its own plasmid
The first task is to identify which bacterial cells have taken up the plasmid. One way to do this is to use the fact that bacteria have evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium. The genes for the production of these enzymes are found in the plasmids. Some plasmids carry genes for resistance to more than one antibiotic.
The task of finding out which bacterial cells have taken up which plasmids entails using the gene for antibiotic resistance, which is unaffected by the introduction of the new gene. The process using ampicillin works as follows:
* All the bacterial cells are grown on a medium that contains the antibiotic ampicillin
* Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
* These bacterial cells are able to break down the ampicillin and therefore survive
* The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die
However some cells will have taken up the plasmids which closed up without incorporating the new gene and these cells will also have survived. The next task is to identify the cells without the new gene and eliminate them. This is achieved using marker genes
Marker Genes
- marker genes are used to identify the cells without the new gene and eliminate them. Number of different ways of using marker genes to identify whether a gene has been taken up by a bacterial cell. They all involve using a second, separate gene on the plasmid. This second gene is easily identifiable for one reason or another. For example:
- may be resistant to an antibiotic
- may make a fluorescent protein that is easily seen
- May produce an enzyme whose action can be identified
The use of antibiotic-resistance genes as markers is rather old technology and has been superseded by other methods
To identify those cells with plasmids that have taken up the new gene we use replica plating
This process uses the other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene. As this gene has been cut, no longer produce enzyme that breaks down antibiotic. Therefore can identify these bacteria by growing them on a culture that contains the antibiotic they are no longer resistant to
The problem is that treatment will destroy the very cells that contain the required gene. However by using replica plating it is possible to identify living colonies of bacteria containing the required gene
Called replica plating as use sterile velvet block to create a stamp of the growth media which can then be transferred to different plates- so then transferred to plate with antibiotic after stamp has been made
fluorescent markers
- more rapid method is the transfer of a gene from a jellyfish into the plasmid. The gene in question produces a green fluorescent protein (GFP). The gene to be cloned is transplanted into the centre of the GFP gene. Any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP, but bacterial cells that havnt taken up the gene will continue to produce GFP and fluoresce. As the cells with the desired gene arent killed there is no need for replica plating, results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce. This makes the process more rapid
Enzyme markers
Another gene marker is the gene that produces the enzyme lactase. Lactase will turn a particular colourless substrate blue. Again, the required gene is transplanted into the gene that makes lactase.
If a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase. Therefore, when these bacterial cells are grown on the colourless substrate the will be unable to change its colour. Where the gene hasn’t been taken up by bacteria, they will not turn the substrate blue. These bacteria can be discounted
What happens after cells containing recombinant DNA have been identified
-GROW/CLONE host cell
- a fermenter is used to grow multiple copies of the host cell which have been identified as containing the recombinant plasmid
- This large, cloned population of the host cell can then produce the protein coded for by the inserted DNA fragment eg bacteria producing insulin from the inserted insulin gene
Polymerase chain reaction ‘ingrediants’
Involved amplifying DNA fragments in vitro- means done outside a living organism on an automated machine
Requires:
* DNA fragment that is being amplified
* DNA polymerase- taq polymerase- special type that doesnt denature under high temperatures as is obtained from bacteria in hot springs
* Primers- short sequences of nucleotides that have a set of bases complementary to those at one end of the two DNA fragments
* Nucleotides- which contain each of the four bases found in DNA
* thermocycler- a computer controlled machine that varies temperatures over a precise period of time
Steps of PCR
- Separation of the DNA strand. DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. Temperature increased to 95’C, causing the two strands of DNA fragment to separate due to breaking of the hydrogen bonds between the two stands
- Addition (annealing) of the primers. Mixture cooled to 55’C, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment. Primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from re-joining
- Synthesis of DNA. Temperature increased to 72’C. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.
Now two copies of the original DNA fragment
advantages of invitro gene cloning
- rapid/automated/more efficient. Particularly valuable when only small amount of DNA available, eg at a crime scene. This can be quickly increased so no lost valuable time eg for forensic analysis
- doesnt require living cells. No complex culturing techniques required
However, may increase/ keep producing any DNA that has contaminated the sample- errors copied into subsequent samples
advantages of in vivo gene cloning
- useful when we wish to introduce a gene to another organism. As it involves vectors, plasmid can be used to deliver the gene into another organism eg a human. Done through gene therapy
-No contamination risk. Gene has been cut by same restriction endonuclease that will match sticky ends of plasmid so contaminant DNA wont be taken up by the plasmid - Very accurate- very few errors
- Cuts out specific genes- very precise as produces many samples of specific genes not just DNA sample
- Produces transformed bacteria that can be used to produce large quantities of gene products eg making insulin
Benefits of recombinant DNA technology
- microorganisms can be modified to produce a range of substances, eg antibiotics, hormones and enzymes, that are used to treat diseases and disorders
-microorganisms can be used to control pollution eg to break up and digest oil slicks or destroy harmful gases from factories. Need to ensure doesnt destroy oil in places where it is required like a car engine, to do this a suicide gene can be incorporated that causes the bacteria to destroy themselves once the oil slick has been digested - genetically modified plants can be transformed to produce a specific substance in a particular organ of the plant eg manufacturing antibiotics
- genetically modified crops can be engineered to have financial and environmental advantages. These include making plants more tolerant to environmental extremes
- genetically modified crops can help prevent certain diseases eg golden rice and vitamin A
- GM animals are able to produce expensive drugs, antibiotics, hormones and enzymes relatively cheaply
- replacing defective genes might be used to cure certain genetic disorders eg cystic fibrosis
- ## genetic fingerprinting used in forensic science
Risks of recombinant DNA technology
- impossible to predict with complete accuracy what the ecological consequences of releasing GMO into the environment will be
- recombinant gene may pass from the organism it was placed into a completely different one
- any manipulation of DNA of a cell will have consequences for the metabolic pathways within that cell.
- GM bacteria often have antibiotic resistant marker genes that have been added. Might spread the antibiotic resistance to harmful bacteria
- All genes mutate, might have severe consequences if GM bacteria mutates
- may be long term consequences of introducing new gene combinations
- financial consequences
- could be taken to far eg replacing genes for intelligence, body type, different facial features etc
- could get into wrong hands eg use to control opposition
- high financial cost
- genetic fingerprinting could be misused eg exchanging someone’s DNA sample
- may think immoral to tamper with genes at all
- is it right that an individual or company can effectively own a gene
What is a DNA probe
- short, single-stranded length of DNA that has some sort of label attached that makes it easily identifiable.
- used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks. Can be used to screen for potential genetic disorder or for the presence of cancer causing oncogenes. Possible to screen for multiple diseases simultaneously using a microarray, where multiple different DNA probes are attached
- created to have a complmentary base sequence to the allele being screened for. The patients DNA sample is treated to make it single stranded and it is then mixed with the DNA probes
- if the patient has the allele, then the DNA probe will hybridise and the label indicates the presence
two most commonly used probes
- radioactively labelled probes, made up of nucleotides with the isotope 32P. The probe is identified using an X-ray film that is exposed by radioactivity
-fluorescently labelled probes. Emit light (fluoresce) under certain conditions, for instance when the probe has bound to the target DNA sequence
how are DNA probes used to identify particular alleles of genes
- base sequences are complementary to the part of the base sequence of DNA that make up the allele of the gene we want to find
- the double-stranded DNA that is being tested is treated to separate its two strands
-the separated DNA strands are mixed with the probe, which binds to the complementary base sequence on one of the strands. This is known as DNA hybridisation - The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits
Before we make the specific probe we need to know the base sequence in the particular allele we are trying to locate. A number of different methods are used to sequence the exact order of bases in a length of DNA
DNA hybridisation
- takes place when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases. Before hybridisation can take place, the two strands of the DNA molecule must be separated.
- achieved by heating DNA until its double strand separates into its two complementary single strands (denaturation). When cooled, the complementary bases on each strand recombine (anneal) with each other to reform the original double strand.
Given sufficient time, all strands in a mixture will pair up with their partners. If however, other complementary sections of DNA are present in the mixture as the DNA cools, these are just as likely to anneal with one of the separated DNA strands as the two strands are with one another
locating specific alleles of genes
- determine the sequence of nucleotide bases of the mutant allele we are trying to locate. Can be achieved using DNA sequencing techniques. Now have extensive genetic libraries that store the base sequences of most genetic diseases and so we can simply refer to these to obtain the sequence
- A fragment of DNA is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate. This is done through the gene machine and will be amplified using PCR
- The DNA probe is made by attaching a marker to the DNA fragment eg a fluorescent dye or radioactive label
- The DNA from the person suspected of having the mutant allele will be heated/ undergo DNA hybridisation to seperate its two strands
- Seperated strands are cooled in a mixture containing many of the DNA probes
- If DNA contains mutant/ desired allele, one of the probes will bind
- DNA is washed clean of any unattached probes
- Remaining hybridised DNA will now be fluorescently labelled with the dye attached to the probe
- The dye is detected by shining light onto the fragments causing the dye to fluoresce which can be seen using a special microscope or use X-rays to detect for radioactively labelled probes
genetic screening
- many genetic disorders eg sickle-cell anemia, are the result of gene mutations. Gene mutations may arrise if one or more nucleotide bases in DNA are changed in any one of a variety of ways.
- important to screen individuals who may carry a mutant allele. Such individuals often have a family history if a disease and screening can determine the probabilities of a couple having offspring with a genetic disorder. As a result, potential parents who are at risk can obtain advice from a genetic counsellor about the implications of having children, based on their family history and the results of the genetic screening
- possible to fix hundreds of different DNA probes in an array (pattern) on a glass slide. By adding a sample of DNA to the array, any complementary DNA sequences in the donor will bind to one or more probes. Possible to test simultaneously for many genetic disorders by detecting fluorescnece that occurs where binding has taken place
- Valuable in detection of oncogenes. Cancers develop as a result of mutations that prevent tumour suppressor genes inhibiting cell division. Mutations of both alleles must be present to inactivate the tumour suppressor genes and to initiate the development of a tumour. Some people inherit one mutated tumour supressor gene and these individuals have a greater risk of developing cancer.
- If a mutated gene is detected by genetic screening, individuals can make informed decisions about their lifestyle and future treatment. Can give up smoking, lose weight, eat more healthily and avoid mutagens as far as possible. Also can regularly check themselves for early signs of cancer which can lead to an early diagnosis and better change of successful treatmwnt. May chose to undergo some form of surgery or other treatment
personalised medicine
- one of the advantages of genetic screening
- allows Drs to provide advice and health care based on an individuals genotype. Some peoples genes can mean that a particular drug may be either more or less effective in treating a condition. By genetically screenng the dose of the drug to produce a desired outcome can be determined more exactly. Can save many that could be spent on overprescribing the drug and avoids medications that could cause harm/ false hope
- eg for people with diabetes vitamin E may reduce their risk of CVD but with other geneotypes can actually increase the risk
Genetic counselling
- social work where advice and info are given that enable people to make personal decisions about themselves or their offspring. One imporant aspect is to research family history of an inherited disease and to advice parents on the likelihood of it arrising in their children
- Can inform pepople of emotional, psychological, medical, social and economic consequences of a disease to allow couples to make decisions about having children. May make aware of additional medical tests that give more accurate prediction eg IVF screening of embryos
- Genetic screening provides genetic counsellor with bases for informed discussion
Genetic fingerprinting
- relies on the fact that the genome of most eukaryotic organisms contains many repetitive, non-coding bases of DNA. DNA bases which are non-coding are known as variable number tandem repeats (VNTRs). For every individual the number and length of VNTRs has a unique pattern. They are different in all individuals except identical twins and the probability of two individuals having identical sequences of these VNTRs is extremly small.
- The more closely related two individuals are, the more similar the VNTRs will be
Gel electrophoresis
- used to separate DNA fragments according to their size. Placed on to an agar gel and voltage applied across it
- Resistance of the gel means the larger the fragments, the more slowly they move. Therfore, over a fixed period, the smaller fragments move futher than the larger ones. In this way DNA fragments of different lengths are seperated.
- If the DNA fragments are labelled eg with radioactive DNA probes, their final positions in te gel can be determined by placing a sheet of X-ray film over the agar gel for several hours. The radioactivity from each DNA fragment exposes the film and shows where the fragment is situated on the gel
- Only DNA fragments up to around 500 bases long can be sequenced in this way. Larger genes and whole genomes must therefore be cut into smaller fragments by restriction endonucleases
Method of genetic fingerprinting
- Extraction. Tiny samply of DNA is enough to give genetic fingerprint. Extract DNA by seperating it from the rest of the cell. Then if amount is smaller increase the quantity using PCR
- Digestion, Cut into fragments using the same restriction endonucleases. Chosen for their ability to cut close to, but not within, target DNA
- Separation. Fragments of DNA are seperated according to size by gel elctrophoresis under influence of electrical voltage. Then immersed in alkali in order to seperate the double strands into single strands
- Hybridisation. Radioactive or fluorescent DNA probes are now used to bind with VNTRs. Probes have base sequence which are complementary to the base sequences of VNTRs, and bind to them under specific conditions. Process carried out with different probes, which bind to different target DNA sequences
- Development. An X-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes (if using fluorescnet probes the positions are located visually). Because these points correspond to the position of the DNA fragments as seperated during electrophoresis, a series of bars is revealed. The pattern of the bands is unique to every individual except identical twins
DNA fingerprints are visually checked. If there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of DNA fragments from the bands. Does this using data obtained by measuring the distances travelled using electrophoresis by known lengths of DNA. Finally, the odds are calculated of someone else having an identical fingerprint. Closer the match between the two patterns, greater the probability that the two sets of DNA have come from the same person
Uses of DNA fingerprinting
- Genetic relationships and variability. Help resolve questions of paternity. Each bond on a DNA fingerprint of an individual should have a corresponding band in one of the parents DNA fingerprint. A population whose members have very similar genetic fingerprints has little genetic diversity
- Forensic science. Can establish whether a person is present at the scene of a crime. Important to remember DNA may belong to a very close relative, DNA may be contaminated after the crime- by suspects DNA or by chemicals that affected the action of the restriction endonucleases used in preparing the fingerprint
- Medical diagnosis. Help in diagnosing diseases such as Huntington’s disease- results from three base sequence AGC at one end of a gene on chromosome 4 being repeated over and over again. Sample of DNA from a person with the allele for Huntington’s disease can be cut with restriction endonucleases and a DNA fingerprint prepared. This can then be matched with fingerprints of people with various forms of the disease and those without the disease, in this way the probability of developing the symptoms and when can be determined. Genetic fingerprints are used to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with that of known pathogens
- Plant and animal breeding. Used to prevent undesirable interbreeding during breeding programmes on farms or in zoos. Can also identify plants or animals that have a particular allele of a desirable gene. Individuals with this allele can be selected for breeding in order to increase the probability of their offspring having the characteristic that it produces. Another application is the determination of paternity in animals and thus establishing the pedigree/ family tree of an individual
