Topic 8: Recombinant DNA Flashcards
What is recombinant DNA?
The introduction of a foreign gene into the DNA of another organism.
The resulting organism is a genetically modified organism (GMO)
Why does DNA still function normally when it is transferred into another organism?
The genetic code is universal, and so are the mechanisms of transcription and evolution translation, so the same proteins are made by the same DNA, regardless of species.
This is indirect evidence for evolution
What are the steps for making a protein using recombinant DNA technology?
- Isolation - of DNA fragments that have the gene for the desired protein
- Insertion - of DNA fragment into a vector
- Transformation - inserting vector with DNA into suitable host cells
- Identification - of host cells that have taken up the gene using gene markers
- Growth / cloning - of host cell population
What are the three methods of isolating DNA fragments?
- Using reverse transcriptase
- Using restriction endonucleases
- Gene machine
Describe how you could use reverse transcriptase to isolate DNA fragments
- A cell that readily produces the protein is selected
- These cells have large quantities of the mRNA needed, so it is easier to extract
- Reverse transcriptase is used to produce a single strand of complementary DNA (cDNA) to the mRNA
- DNA polymerase is used to form the other strand using free complementary nucleotides
Describe how you could use restriction endonucleases to isolate DNA fragments
Restriction endonucleases are enzymes found in bacteria that cut DNA at specific base sequences (recognition sequences) as a defence mechanism against viruses.
There are 2 ways they can cut DNA:
- Blunt ends - the cut occurs between 2 opposite base pairs, leaving 2 straight edges on the molecule.
- Sticky ends - enzyme cuts DNA in a staggered fashion, leaving an uneven cut where each strand of the DNA has exposed, unpaired bases. These can attach to complementary bases of other sticky ends if the have been cut by the same enzyme. Occurs with palindromic recognition sequences.
What is a palindromic recognition sequence?
The sequence and its complement are the same but reversed, between 4 and 8 bases long
Describe how you would use the gene machine to isolate DNA fragments
Manufactures genes in a laboratory.
- Desired nucleotide sequence derived from target protein (protein = amino acids = mRNA = DNA)
- Sequence fed in computer and checked for biosafety
- Oligonucleotides designed and produced (small, overlapping single nucleotide strands) that assemble into the complete gene.
- The gene created from
oligonucleotides doesn’t have introns or non-coding DNA - Gene is replicated many times using Polymerase Chain Reaction (PCR), which also makes it double stranded
- Using sticky ends, gene is inserted into a bacterial plasmid (vector)
What are the advantages of the gene machine?
- Fast
- Any sequence can be produced
- High accuracy
- Can remove introns so can be transcribed and translated by prokaryotic cells
How does in vivo gene cloning work?
If DNA from different organisms is cut using the same restriction endonuclease, the 2 fragments will have complementary sticky ends. Once the bases have formed their hydrogen bonds, DNA ligase is used to join the sugar-phosphate backbones
What are the steps to in vivo gene cloning?
- Preparing DNA fragment for insertion
- Insertion of genes into a vector
- Transformation - introducing recombinant plasmids to host cells
- Identifying which cells have taken up recombinant plasmids
How are DNA fragments prepared for insertion in in vivo gene cloning?
Extra lengths of DNA must be added to the fragment.
- Promoter - a length of DNA before the fragment, allowing RNA polymerase and transcriptional factors to bind to initiate transcription
- Terminator - a length of DNA after the fragment, which releases RNA polymerase to stop transcription
How does insertion of genes into a vector in in vivo gene cloning work?
The most common vector is a plasmid. They almost always contain antibiotic resistance genes, and restriction endonucleases are used to break the plasmid loop at one of those genes. The same endonuclease is used as the one that cut the fragment, so the sticky ends are complementary. The fragments are mixed with the open plasmids and joined by DNA ligase.
Describe the transformation stage of in vivo gene cloning
- Plasmids are mixed with bacterial cells in a medium containing Ca2+ ions and heat shock (42 degrees) is applied for 2 mins, both to increase bacterial cell wall permeability, allowing plasmids to pass through.
- Not all bacterial cells will have the recombinant DNA:
- Less than 1% of bacteria take up the recombinant plasmids
- Some plasmids self-ligate and close up before incorporating the DNA fragment
- Sometimes DNA fragment ends join together to form its own plasmid.
How do we identify which bacteria have taken up any kind of plasmid in in vivo gene cloning?
Many plasmids have antibiotic resistance genes, e.g for ampicillin. All the bacterial cells are grown on a medium containing ampicillin.
Bacterial cells that have taken up the plasmids have acquired the ampicillin resistance gene, so survive, and those that haven’t taken up a plasmid die.
However, not all plasmids taken up will be recombinant, so can’t identify which have the target gene.
How do we identify which bacterial cells have taken up the recombinant plasmids in in vivo cloning?
Using marker genes:
- Antibiotic-resistance markers
- Fluorescent markers
- Enzyme markers
How are antibiotic-resistance markers used to determine which bacteria have taken up recombinant plasmids in in vivo gene cloning?
- Target gene is inserted into the resistance gene of antibiotic A in a plasmid which also has a resistance gene of antibiotic B (so it is no longer resistant to A)
- Bacteria are cultured in agar with antibiotic B - those with no plasmid die
- Replica plating uses a pad to make a copy of the cultures on this agar
- Replica plate is treated with antibiotic A - colonies that die are recombinant
How are fluorescent markers used to determine which bacteria have taken up recombinant plasmids in in vivo gene cloning?
- Organisms like jellyfish produce fluorescent proteins whose genes are inserted into a plasmid.
- Target gene is inserted into the fluorescence gene - plasmid also has (ampicillin) resistance gene.
- Transfer plasmids to bacterial cells and grow in agar containing ampicillin.
- Bacterial cells with no plasmids are killed, those with self-ligated plasmids fluoresce, and living bacteria with no fluorescence have the recombinant DNA.
How are enzyme markers used to determine which bacteria have taken up recombinant plasmids in in vivo gene cloning?
- There is a particular substrate that lactase turns from colourless to blue
- Insert target gene into the lactase gene in a plasmid with an ampicillin resistance gene
- Transfer plasmids into bacterial cells and grow in agar containing ampicillin and substrate
- Bacteria with no plasmids are killed, those with self-ligated plasmids turn the substrate blue, those with recombinant plasmids will remain colourless
What does in vivo and in vitro mean?
In vivo = performed in a living organism
In vitro = performed in a test tube / culture dish
What is PCR?
The Polymerase Chain Reaction - a method of copying / amplifying DNA fragments in the lab.
An automated process, so rapid and efficient. Provides a sufficient mass of DNA for forensic analysis and genetic fingerprinting.
What does the Polymerase Chain Reaction need to work?
- DNA fragment to be copied
- (taq) DNA polymerase - joins thousands of nucleotides per minute. taq polymerase is found from bacteria in hot springs, so is thermostable (heat-tolerant so doesn’t denature in the high temps needed)
- Primers - short, single-stranded DNA with a complementary base sequence to the start of the DNA fragment. Allows attachment of DNA polymerase and prevents 2 DNA strands rejoining
- Free nucleotides
- Thermocycler - computer-controlled machine that varies temperature precisely over time
Describe the stages of PCR
- Separation of DNA strand: DNA fragments, primers, DNA polymerase are placed in thermocycler. Heated to 95 degrees, breaking hydrogen bonds between bases and separating strands
- Addition (Annealing) of primers: cooled to 55 degrees, so primers join to complementary bases at end of DNA fragment. Provide starting sequence for new strand as DNA polymerase can only attach nucleotides to the end of an existing chain
- DNA synthesis: temperature increased to 72 degrees - optimum temperature for taq DNA polymerase to synthesise the new strand
- Cycle is repeated many times. With each cycle, mass of DNA doubles. One cycle takes 2 mins, and over 1 million copies can be made in 25 cycles (50 mins)
What are the advantages of in vitro cloning?
- Extremely rapid - valuable when only a minute amount of DNA is available, however PCR massively increases any contaminating DNA
- Doesn’t require living cells - only needs the target sequence, no complex and time consuming culturing techniques needed
What are the advantages of in vivo cloning?
- Useful when introducing a gene into another organism
- Almost no risk of contamination - contaminant DNA won’t be taken up by the plasmid as it hasn’t been cut by the same restriction endonuclease
- Very accurate - DNA copied has few, if any, errors as mutations are rare. In PCR any errors / contaminants will also be copied in subsequent cycles
- Cuts out specific genes - precise procedure as it doesn’t copy the whole DNA sample
- Produces transformed bacteria that can produce large quantities of gene products
What are the benefits of recombinant DNA technology?
- GM microorganisms can produce antibiotics, hormones etc to treat disease
- GM microorganisms can control pollution e.g destroying oil slicks / harmful gases
- GM plants can produce substances like drugs and antigens
- GM crops can have financial / environmental advantages e.g drought, heat, cold, salt resistance
- GM crops can help prevent certain diseases, e.g having certain vitamins
- GM animals can produce expensive drugs, hormones, enzymes cheaply
- Gene therapy can cure disorders e.g cystic fibrosis
- Genetic fingerprinting can be used in forensic science
What are the risks of recombinant DNA technology?
- Impossible to predict ecological consequences of releasing GMOs
- Recombinant gene may pass into unintended organisms
- Any manipulation of DNA in cells can affect metabolic pathways
- What are the consequences of it mutating?
- Long term consequences on future evolution?
- Financial consequences of developing plants / animals to grow in new regions?
- Could it lead to eugenics?
- Is the financial cost justified?
- Could the ability to manipulate genes get into the wrong hands?
- Could genetic fingerprinting lead to framing people for crimes?
- Is it immoral to tamper with genes?
- Is it right that a company can patent a gene?
What is gene therapy?
The replacement of defective genes by the introduction of healthy genes.
Process of putting a corrected gene into a chromosome = transfection. The cell that has received the new gene is transfected.
What are the 2 approaches to gene therapy?
- Somatic cell therapy
- Germ-line therapy
What is somatic cell therapy in gene therapy?
Targets the affected tissues. Not passed on to future generations. Long-term goal is to target stem cells so treatment is effective for the patient’s lifetime
What is germ-line therapy in gene therapy?
Replacing the defective gene with a healthy gene or supplementing with a dominant allele if it is a recessive disorder, in a fertilised egg.
All offspring cells develop normally, so is passed on to future generations
What are DNA probes?
Short (up to 20 bases), single-stranded lengths of DNA, with a base sequence complementary to part of a target gene.
Easily identifiable as they are labelled radioactively (nucleotides with 32P isotopes detected with x-ray film) or fluorescently.
Probes are designed complementary to (mutant) allele from genetic library, then made with PCR.
What is DNA hybridisation?
- Occurs when a section of DNA / RNA is combined with a single-stranded section of DNA with complementary bases.
- DNA is heated until its strands separate (denaturation).
- DNA probes are added and allowed to cool.
- Probe will bind to complementary section of DNA (annealing).
- DNA is washed to remove unattached probe and the bound probe is detected
How do we locate specific alleles on a length of DNA?
- DNA sequencing / genetic libraries used to find sequence of target mutant allele
- Fragments of complementary DNA probes produced and amplified using PCR, with markers attached (radioactivity / fluorescence)
- DNA from patient is heated to separate strands, then cooled with the probes
- If DNA has the mutant allele, the probes will bind
- DNA is washed clean of unattached probes, so hybridised DNA can be detected
Why is genetic screening important?
It is important to screen individuals who may have a mutant / disease-causing allele e.g if they have a family history of disease.
Can be used to determine the probability of a couple having offspring with the genetic disorder.
It is also valuable for the detection of oncogenes and mutated tumour suppressor genes, to find individuals at a greater risk of cancer, so they can make informed decisions about their lifestyle, future treatment, and to screen for cancer early and regularly
How does genetic screening work?
To screen for disorders, hundreds of different DNA probes for different disorders are fixed into wells on a microarray slide. By adding sample DNA to the slide, any complementary sequences will bind to the probes. It is possible to test for many disorders by detecting fluorescence that occurs when binding occurs.
What is personalised medicine?
Genetic screening allows doctors to provide healthcare based on a patient’s genotype.
Doctors can determine the dose of the drug that would be the most effective, saving money from overprescription, avoiding medication that could cause harm and preventing false hope.
What is genetic counselling?
A form of social work, where advice and information are given that enable people to make personal decisions about themselves and their offspring, e.g to research family history of an inherited disease and advise the parents on the likelihood of it arising in their children.
Can also inform the couple of the medical, psychological, social and economic effects of the disease, and make them aware of any further tests that might give a more accurate prediction of their offspring’s condition, e.g IVF screening of embryos
What are VNTRs?
The genome of most eukaryotic organisms is mostly non-coding DNA. These sections are Variable Number Tandem Repeats (VNTRs).
For every individual, the numbers and lengths of VNTRs have a unique pattern, apart from identical twins. The more closely related two individuals are, the more similar their VNTRs will be.
What is gel electrophoresis and how does it work?
Used to separate DNA fragments based on size.
Fragments are placed in wells on the gel and a voltage is applied (negative DNA moves away from the negative charge).
Resistance of the gel means larger fragments move slower, and so less far, and get separated.
If the DNA fragments are labelled, their final positions on the gel can be detected. Whole genomes have to be cut into fragments by restriction endonucleases.
What are the steps to genetic fingerprinting?
- Extraction
- Digestion
- Separation
- Hybridisation
- Development
Describe the extraction step in genetic fingerprinting
Cells from a sample of blood, hair, skin etc are broken down and DNA is extracted and amplified with PCR.
Describe the digestion step in genetic fingerprinting
DNA is cut into fragments with the same restriction endonucleases, which are chosen for their ability to cut close to, but not within VNTRs
Describe the separation stage in genetic fingerprinting
Gel electrophoresis is used to separate strands by size. Gel is immersed in alkali to separate the double strands into single-stranded molecules.
Describe the hybridisation stage in genetic fingerprinting
DNA on gel is transferred to a nylon membrane. DNA probes bind to the complementary VNTRs. Different probes can be used to bind to different DNA sequences.
Describe the development stage in genetic fingerprinting
X-ray film is put over the nylon membrane and radioactive probes on DNA expose the film, producing a visible pattern of bands, which is unique to every individual except twins.
How are the results of DNA fingerprints interpreted?
To interpret results, DNA fingerprints are visually checked.
If a match is suspected, the patterns of bars are passed through an automated scanning machine, which calculates lengths of fragments using distances travelled by known lengths of DNA.
The closer the match between the 2 patterns, the greater the probability the DNA came from the same person
What are some uses of genetic fingerprinting?
- Determining genetic relationships
- Forensic science
- Medical diagnosis
- Breeding programmes
Describe how genetic fingerprinting is used to determine relationships between individuals
e.g paternity testing.
Each band on the DNA fingerprint of a child should correspond with a band on one of the parents’ fingerprints.
Can also determine the genetic diversity of a population
Describe how genetic fingerprinting can be used in forensic science
DNA is often left at crime scenes, and fingerprinting can be used to establish whether a person was likely present.
However, DNA may have been left on another occasion, DNA may be from a close relative, DNA sample may have been contaminated.
The probability that someone else’s DNA may match the suspect is then calculated
Describe how genetic fingerprinting can be used in medical diagnosis
Can help diagnose some diseases, e.g Huntington’s disease.
People with certain lengths / numbers of these sequences are more likely to develop symptoms of the disease.
Can also be used to identify the nature of a microbial infection by comparing the fingerprint of the patient’s microbe with that of known pathogens.
Describe how genetic fingerprinting can be used in breeding programmes
Can be used to prevent inbreeding in farms and zoos.
Can also identify individuals with desirable alleles, so they can be selected for breeding.
Can also be used to determine paternity of animals, so establishing the family tree.
