chapter 21 - recombinant DNA technology Flashcards
what is recombinant DNA
DNA of two different organisms that has been combined. isolate genes. clone genes. and transfer them into micro organisms.
what is an organism formed from recombinant DNA called.
known as a transgenic / genetically modified organism (GMO)
why is possible that DNA of one organism is accepted by a different species but also functions normally when it is transferred
because the genetic code is the same in all organisms. it is universal. making the proteins is also universal as the mechanisms of transcription and translation are essentially the same in all organisms. therefore transferred DNA can be transcribed and translated within the cells of the recipient (transgenic organism)
describe the process of 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- transfer of DNA into a suitable host cell
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
What is the meaning of sequencing a genome
Working out all the DNA base sequences for all of the DNA in the cell
How long did the human genome take to complete
13 years
Give an example of a method that is used to sequence a genome
The sanger method
We do not specifically need to know the methods for AQA, however the methods are continuously being improved and updated. At this current moment they are mostly automated.
Why is it easier to work out the proteome for prokaryotes
because prokaryotes contain no introns. Therefore the genome is used directly to work out the proteome. This is useful to create vaccines as the antigens can be worked out directly.
Why is it harder to translate the proteome in eukaryotes.
Eukaryotes have Intron’s and regulatory genes in DNA. Therefore the genome cannot be easily used to translate the proteome.
Why is recombinant DNA technology used
It is used to improve industrial processes and medical treatment
What are three methods to creating fragments of DNA during isolation
- conversion of mRNA to cDNA using reverse transcriptase.
- Using restriction endonucleases to splice out desired genes from DNA.
- Using a gene machine to create desired genes [based on protein structure]
What are retroviruses
They are a group of viruses [HIV]
Describe the process of using reverse transcriptase to isolate a gene
- A cell that naturally produces the proteins of interest is selected
- beta cells in the islets of Langerhans in the human pancreas are specialized to produce insulin, they make a lot of mRNA that codes for insulin.
- Messenger RNA code for insulin
- MRNA acts as a template on which a single stranded complementary copy of DNA is formed.
- reverse transcriptase joins complementary DNA nucleotides and cDNA is formed [single-stranded]
- cDNA is isolated via the hydrolysis of mRNA with an enzyme
- double stranded DNA is formed on the template of the sea DNA using DNA polymerase.
- A copy of the human insulin gene is made
What is the advantage of cDNA
It is intron free is it is based on the mRNA template
What does cDNA stand for
Complementary DNA
What are restriction endonucleases
They are enzymes that cut up DNA
Where are restriction endonucleases found
They naturally occur in bacteria as a defense mechanism
What is the site called by restriction endonucleases cut
The recognition sites/sequence
What type of ends are left when restriction endonucleases cut at the same location on DNA
Blunt ends
What type of ends are left one restriction endonucleases cut in a staggered fashion
Sticky ends
what are exposed staggered ends called
Palindromic/palindromic sequence
What is the importance of sticky ends
They have the ability to join to DNA with complementary base pairs
What does a palindromic sequence mean
It’s reads the same forwards as it does backwards
Which type of restriction endonuclease is more useful [one that produces a palindromic sequence/sticky ends or one that produces blunt ends]
One that produces a palindromic sequence/sticky ends.
When this section of DNA is placed into a new organism it’s can more easily combine with the original DNA of that organism.
It makes it easier to join the DNA with the next sample
Describe the process of creating a DNA fragments via a gene machine
Scientist would examine the protein of interest and determine the primary structure [amino acid sequence] from this they were determined the mRNA sequence and then the DNA sequence that follows.
The DNA sequence is entered into a computer and the sequence is checked for biosafety and bio security to ensure it meets international standards as well as various ethical requirements.
The computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides. Which can be assembled into the desired genes.

next, 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 join together to make the desired gene. The stream does not contain introns or non-coding DNA.
The chain is replicated using the polymerase chain reaction [PCR]
The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double strand of gene. Using sticky ends the gene can be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to another organism in the future the jeans are checked using standard sequencing techniques and those with errors are rejected.
What is the advantage of producing a DNA fragments by the gene machine
That any sequence of nucleotides can be produced in a very short time [as little as 10 days] and with great accuracy
a further advantage is that these artificial genes are also free of introns and other non-coding data that’s all they can be transcribed and translated by prokaryotic cells.
What are the advantages of using reverse transcriptase
The mRNA from a cell has been actively transcribed, the availability of mRNA is used to make cDNA.
What are the disadvantages of using reverse transcriptase
It is more time-consuming therefore it is more technical difficult
What are the advantages of using restriction endonucleases
Sticky ends on a DNA fragments make it easier to insert and make it easier to make recombinant DNA
What are the disadvantages of using restriction endonucleases
it still contains introns
What are The advantages of using a gene machine
You can design the exact DNA fragments required with sticky ends and preferential codons.
What is the disadvantage of using a gene machine
You need to know the amino acid sequence/primary structure/sequence
What is in vivo
It is transferring the fragments to a host cell using a vector
What is in vitro
Using the polymerase chain reaction [PCR]
Describe the process of using sticky ends to combine DNA from different sources
The DNA double helix is cut at the recognition site with restriction endonuclease. DNA from another source cut with the same restriction endonuclease Joins with the other DNA fragment via DNA ligase which joins the two sections forming recombinant DNA.
What is the function of DNA ligase
It is used to bind the phosphate sugar framework of the two sections of DNA to form recombinant DNA. Sticky ends have considerable importance because, provided the same restriction endonucleases used. We can combine the DNA of one organism with that of any other organism.
how do we prepare the DNA fragments for incision (in vivo)
we add a promoter region - To the start of the DNA fragment, this is a sequence of DNA which is the binding site for DNA polymerase to enable transcription to occur.
We also add a terminator region - To the end of a gene/fragment. Its causes RNA polymerase to detach and stop transcription. So only one gene at a time can be copied into mRNA.
What is a vector
Something that can carry the isolated DNA fragment into the host cell
What are the most common types of vector
Plasmids [circular DNA, separate from the main bacterial genome which only contains a few genes.]
Describe the process of incision of a DNA fragment into a factor
The plasmid is cut open using the same restriction endonuclease
therefore the same sticky ends are formed
DNA fragments sticky ends/expose nucleotides are complementary to the sticky ends on the plasmid
Therefore the DNA fragment is now attached to the plasmid
DNA ligase sticks plasmid and the DNA fragment together [Annealing them]
DNA ligase catalyzes a condensation reaction to form phosphodiester bonds between nucleotides.
Describe the overall process of in vivo Cloning
create DNA fragments of the gene of interest
Insert DNA fragments into a vector
Transform the host cell with a vector
Identify the transformed cells
Go to the host cell, clone/make copies of the gene.
What does transformation mean
It means getting a plasmid inside the bacteria/host cell. the gene will be expressed on the Protein will be made.
How do we insert the plasmid into the host cell
to do this the cell membrane of the host cell must be more permeable
To do this we mix The host cells with calcium ions and we also heat shot this mixture (a so an increase in temperature)
This enables a vector to enter the host cells cytoplasm.
Why won’t all the bacterial cells possess the DNA fragments
Only if you bacterial cells [as few as one percent] take up the plasmids win the two or mixed together
some plasmids will have closed up again without incorporating the DNA fragment
Sometimes the DNA fragment ends join together to form its own plasmid.
How can we identify which bacterial cells have taken up the plasmid
we test with 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 and the genes for the production of these enzymes are found in the plasmid. And some plasmid carry genes for the resistance to more than one antibiotic. The task of finding out which bacterial cells have taken up the plasmid entails using the gene for antibiotic resistance which is unaffected by the introduction of the new gene.
What’s all the three reasons for the bacterial cells not taking up the plasmid
The Recombinant plasmid does not get inside the cell even though calcium ions and temperatures be increased
The plasmid may rejoin it before the DNA fragment enters
The DNA fragments may stick to themselves, Rather than inserting into the plasmid
Why is it important that we identify which bacteria cells have taken up the recombinant plasmid
Because when we produce The bacteria in mass we want to ensure that this bacteria contain the recombinant plasmid
What are the three key methods used to identify which bacteria successfully took up the recombinant plasmid
antibiotic resistance
genes coding for fluorescent proteins
genes coding for enzymes
Describe the process of antibiotic resistance [testing for a recombinant plasmid in a bacterial cell]
The bacterial plasmid is inserted with a tetracycline gene and an ampicillin gene.
The DNA fragments is inserted in the middle of the tetracycline gene purposely.
therefore this gene will no longer be resistant to tetracycline due to the non-functional protein.
We then grow the bacterium on an agar plate
use a sterile velvet block to stamp the original petri dish into another petri dish with ampicillin antibiotic in the agar. Any colonies that grow contain the plasmid but they could contain the original plasmid or the plasmid with the recombinant DNA fragment. Then we use the imprint of this colony and stamp it on another sterile velvet block With tetracycline in the agar. And any colonies that grow from this will contain both the tetracycline gene and the ampicillin gene. [Does not contain the DNA fragment, as the DNA fragments will disrupt the tetracycline gene]
Describe the process of identifying a bacterial cell with a recombinant plasmid via fluorescent Markers
jellyfish contain a jean witch coast to create a green fluorescent protein (GFP)
GFP can be inserted into the bacterial plasmid
The DNA fragment is inserted into the middle of the GFP gene
We then grow the bacteria on agar
And expose the bacteria to UV light. The glowing colonies do not contain the recombinant plasmid. The non-growing colonies do contain the recombinant plasmid is the GFP gene is interrupted.
Identifying a recombinant plasmid in a bacterial cell using enzyme Markers method
Lactase can turn certain substances from colourless to blue
The gene for lactase is inserted into the plasmid
The DNA fragment is inserted into the lactase gene in the plasmid. right in the middle of the lactase gene.
The color changes are observed
Colonies which Can turn colorless substances blue do not contain the recombinant plasmid as the gene is not disrupted. Colonies which cannot turn the colorless substances blue contain the recombinant plasmid as the gene is disrupted.
Describe the process of growing and multiplying a host cell
a fermenter is used to grow multiple copies of the host cell, which have been identified to contain the recombinant plasmid.
A large cloned population of the host cell can produce the protein. Coded for by the inside to DNA fragment. [For example bacteria producing insulin from the inserted insulin gene]
How can amplifying DNA fragments take place
Once the DNA fragments have been isolated they need to be cloned to create large quantities. This can be done in vivo or in vitro
How can DNA fragments be simplified in in vitro
Fragments of DNA can be amplified in Vitro via the polymerase chain reaction [PCR] this is done via an automated machine
What is the equipment list for polymerase chain reaction’s [PCR]
A thermocycler- changes in temperature, They work in a cycle to produce large quantities of DNA
you need the DNA fragment you want to clone
you need DNA polymerase – taq polymerase [this is obtained from bacteria that live in natural hot springs, they have evolved to have an optimum temperature much higher of 72°C and they can resist temperatures up to 100°C]
You need to add Primers - Short sequences of single-stranded DNA that are complementary to the start and the end of the DNA fragment
you need to add DNA nucleotides
What is the advantage of the polymerase chain reaction
It is rapid and efficient
you can get hundreds of billions of copies made in only hours
quicker and less complex techniques are needed
This method does not require living cells
Describe the process of the polymerase chain reaction/PCR method
denaturing - at 95°C this breaks hydrogen bonds between double stranded DNA, this happens in the thermocycler.

addition/annealing of Primers - The temperature drops to 55°C and this allows primers to attach to the DNA fragment at its ends. This allows DNA polymerase to attach. Hydrogen bonds begin to reform at this temperature, and the primer also helps to prevent separate strands of DNA fragments from simply re-joining.
Synthesis of DNA - DNA polymerase attaches to three nucleotides. it Makes a new strand line next to the template. The temperature is now increased to 72°C for this stage as this is now the optimum temperature for taq DNA polymerase.
What are the advantages of a vivo cloning
It is particularly useful when we wish to introduce a gene into another organism as it involves the use of vectors [gene therapy]
it involves no risk of contamination this is because a gene has been cut by the same restriction endonuclease which can match the sticky ends of the opened up plasmid therefore contaminant DNA will therefore not be taken out by the plasmid.
it is very accurate the DNA copied has very few errors
It’s cuts out specific genes, it is therefore a very precise procedure as the culturing produces many copies of a specific gene and not just copies of the whole DNA sample.
It produces transformed bacteria that can be used to produce large quantities of jean products [for commercial or medical use]
What are DNA probes
They are short single-stranded pieces of DNA that are labeled radioactively/fluorescently so that they can be identified.
What are DNA proves used for
They are used to locate specific alleles of genes and to screen patients of heritable conditions, drug responses or health risks. Therefore DNA probes are created to have a complementary base sequence to the allele on it is being screened for.
describe the process of using DNA probes to identify particular alleles for genes
The DNA probe is made that has a base sequence that is complementary to part of a base sequence of DNA that makes up the alleles of a gene that we want to find.
The double stranded DNA that is being tested is treated two separate it’s two strands.

The separate DNA strands are mixed with the probe which binds to the complementary base sequence on one of the Strands this is known as DNA hybridization
The Site at which the probe binds can be identified by the radioactivity of fluorescent that the probe emits.
What needs to be done before we can make a specific probe
We need to know the base sequence of the particular allele that we are trying to locate. A number of different methods are used to sequence the exact order of bases in a length of DNA.
Describe the process of DNA hybridization
The patient’s DNA sample is heated to make it single-stranded
The heat causes hydrogen bonds between bases to break [denaturing]
The patient single-stranded DNA is mixed with the DNA probe and is cooled to allow any complementary sequences to align and form hydrogen bonds [annealing]
however, some of the patients DNA sample Will anneal back together, but some will anneal with the DNA probe.
How do we locate a specific alleles of a gene
To locate a specific allele, the DNA base sequence must be known to create the DNA probe
This can be determined using sequencing techniques such as the [sanger method]
A fragment of DNA is made in the gene machine
The fragments is then amplified using PCR
A label is added [radioactive nucleotide isotope phosphorus 32 or a fluorescent marker is added] this emits under UV light
The DNA is washed after hypertization so any unbound DNA probes all washed away
The for the presence of fluorescence or radioactive material under an x-ray will show the presence of a DNA probe and therefore the specific allele is located in the patients gene
What is the advantage of using DNA probes to locate specific alleles
It is used to screen for potential genetic disorders or cancer-causing oncogenes
It is possible to screen for multiple diseases simultaneously using an array, when multiple different DNA probes are attached.
Give two advantages of having your DNA screened
you can have personalized medicine, this allows doctors to provide advice and health care based on an individuals genotype.
for genetic counseling, where advice and information is given to enable people to make personalized decisions about themselves or their offspring. One important aspect of genetic counseling is to research the families history of an inherited disease and to advise parents of the likelihood of it arising in their children.
Give an example of why screening your DNA can be advantageous to personalized medicine
Certain drugs such as painkillers or more or less effective depending on your genotype. it’s can also help determine the Best dose Which increases the effectiveness, Safety and can save money
Another example is vitamin E can be given to diabetics to decrease CVD however depending on one’s genotype giving a patient vitamin E could increase CVD.
What is genetic counseling
Having your DNA screened is a decision that must be thought through carefully and must be discussed with patients so they can make an informed decision
Genetic counseling is like a type of social work, Where people can have a family history researchd To consider the likelihood of them carrying any alleles linked to disease For starting a family or for the generational health. Patients are informed of any risks that could affect them or their future offspring.
Give some examples of genetic counseling and what type of diseases they are tested for
Some examples of screening look for jeans associated with or two cystic fibrosis or sickle cell anemia. If there is a family history of breast cancer you may want to be screened for jeans linked to this type of cancer, If you find the allele is present you can be screened for tumors more frequently, or reduce environmental factors or even opt for a mastectomy.
What percentage of human DNA is made up of non-coding DNA/ introns
95%
What are VNTRS
Variable number tandem repeats [found in the introns of human DNA]
what does it mean if two individuals have similar VNTRS
they are more closely related, however it is very unlikely that two individuals will have the same VNTRS
What is genetic fingerprinting overall
it is the analysis of VNTRS DNA fragments, This can be used to determine genetic relationships, genetic variability Within a population
Describe the overall process of genetic fingerprinting overlining the steps
Collection extraction digestion separation hybridization development Analysis
Describe in detail process of genetic fingerprinting
you need to collect and extract your DNA sample even a small sample can be used. As the quantity can be increased by using PCR. Sample can be taken from the blood, hair, body cells
During digestion, restriction endonucleases are added to the DNA sample to cut up the DNA into smaller fragments. you select Restriction endonucleases that are complementary close to the ends of the VNTR fragments of DNA.
during separation the DNA samples are pipetted into a well with agar gel. The gel is placed in a buffer liquid where a voltage is applied. as DNA is negatively charged the DNA will move towards the more positive end of the gel. [Gel electrophoresis]. The agar gel creates resistance as the DNA fragments move in the gel therefore smaller pieces of VNTRS are separated from larger pieces. an alkaline solution is added to separate the double stranded DNA into single strands. (smaller pieces move faster and further through the gel)
DNA probes with markers all added to the gel, hybridization occurs between complementary VNTRS and DNA probes. The DNA probes are made complementary.
during development the agar gel will shrink and crack on the VNTRs and DNA Probes are transferred to a nylon sheet. The nylon sheets are exposed to UV light or x-rays and this shows if DNA probes are present.
During analysis, The position of the DNA bands are compared. To identify genetic relationships, The presence of a disease causing gene and To much unknown samples from a crime scene.


What gives DNA it’s negative charge
The phosphate groups in DNA are negatively charged therefore giving DNA an overall negative charge
What are the three main uses for genetic fingerprinting
- Forensic science to place suspects at a crime scene
- for medical diagnosis
- To ensure animals and plants are not closely related before breeding [this reduces the risk of passing on harmful genetic conditions]
Why do bacteria contain restriction endonucleases?
a bacterium uses Restriction Endonucleases to defend it self against bacterial viruses (bacteriophages/phages). when a phage infects a bacteria is it its DNA replicated. the RE Prevents replication of the phage. by cutting it into many pieces.
What is the function of a primer in PCR
it allows DNA polymerase to attach to the primer and therefore the DNA fragment.
it also prevents DNA fragments from rejoining
uses for gene technology
agriculture
industry
medicine
concerns about recombinant DNA technology
ethical
financial
social
environmental
are the benefits of genetically modified organisms and farming 
The nutritional value of foods gonna be improved
crops can be produced that lack known allergens
GM crops can be engineered to have drought resistant jeans.
Genetically modified crops can produced herbicides to kill pests
genetically modified crops can produce a higher yeild
Genetically modified crops may have a long shelf life
genetically modified crops reduce economical costs
disadvantages of genetically modified organisms in farming
new traits could cause adverse health reactions /(allergens)
crops may limit biodiversity of the local environment (increase competition with native species)
cross pollination could lead to ‘super weeds’
patents restrict farmers from accessing genetically modified seeds
foods with GM components may not be labelled
give examples of genetically modified organisms in farming
Transformed rice contains one gene from Maise and one from soil bacterium. Containing beta-carotene. [our use beta carotene to produce vitamin A]. A deficiency can cause blindness. Up to 500,000 children a year go blind it from vitamin A deficiency.
give another example of GM crops in farming (Bt corn)
Bt corn gene is inserted into the genome. it penetrates and collapsed the cells lining the gut and the insect then dies
in industry what do GMO usually use
enzymes
give an example of GMO in industry (chymosin)
chymosin is a form of cow rennin produced in fungi. the genetically engineered enzyme chymosin can be produced in the fungi faster and more economically in calves.
chymosin can be made into large quantities. relatively cheaply. without killing cows
rennin, also called chymosin, protein-digesting enzyme that curdles milk by transforming caseinogen into insoluble casein; it is found only in the fourth stomach of cud-chewing animals, such as cows. Its action extends the period in which milk is retained in the stomach of the young animal.
give an example of GMOs in medicine (insulin)
insulin treats type 1 diabetes. comes from an animal pancreas
as the insulin wasn’t human it didn’t work as well
human insulin is now made by transformed human micro organisms
benefits of using GMOs in medicine
drugs can be made: quickly, cheaply and in large quantities
this can make medicines more available and more affordable to more people
what are the concerns with the using GMOs in agriculture
farmers may only plant one type of crop. monoculture. ( concerned with diversity )
uncontrollable spread of recombinant DNA with unknown consequences
concerns of GMOs in industry
without proper labeling dome ppl won’t have a choice if they consume gmos or not.
concerns with the purification process of proteins
large biotechnology companies take over forcing smaller companies out of business
concerns with GMOs in medicine
companies limit the use of technology that could save lives
technology can be considered as unethical eg. designer babies
ownership issues
companies can own patents to particular seeds l
what is gene therapy
used to treat human disease
it alters mutated alleles
give an example of a mutated dominant allele
huntingtons disease
Is caused by a frame shift that results in an unstable repeat in the huntingtin gene. (Only 1 mutated dominant allele is needed to induce huntintons). This affects the huntington protein encoded for, leading to a toxic gain of the protein. leading to neurodegeneration
give an example of two mutated recessive alleles. (CF)
cystic fibrosis. (arises from 2 mutated alleles, one from each parent)
3 DNA bases are missing/deleted. Deletion of one amino acid causes (cystic fibrosis trans membrane conductive regulator) CFTR gene to produce a non functional protein. CFTR cannot act as a chloride ion channel protein/(carry out its function in the lungs for example). Chloride ions and water cannot be transported through the ion channels so the epithelial membranes are kept moist as water cannot be transported through them. This is why people with CF tend to have a lot of mucus in their lungs.
how does gene therapy work. CF
the new and correct version of the CFTR gene is placed into the cells of a persons body
the old protein is still coded for
the therapy will only work in cells that receive the therapy
what are some issues / concerns with gene therapy
the effects are short lived.
could induce an immune response
only a small proportion of the genes introduced are expressed
not effective on conditions that arise through multiple genes
what is somatic therapy
altering alleles in the body cells that are most affected by the disorder. only affects those body cells so the disorder may appear in other parts of the body.
what is delivering cloned genes
eg somatic therapy.
the aim of somatic cell gene therapy is to introduce cloned normal genes into the epithelial cells of the lungs
what is gene replacement
when the defective gene is replaced with a healthy gene
what is gene supplementation
in which one or more copies of a healthy gene is added alongside a defective gene. the added genes become dominant alleles. as the recessive alleles are masked
what is germ-line therapy
where alleles in the sex cells are altered. means that every cell in the offspring produces from these cells will be affected by gene therapy. they won’t sufffer from the disease. however, this is currently illegal.
what is a vector
They are used to transfer a desired gene into a host cell.
Plasmids as vectors
small circular rings of double stranded DNA.
They occur naturally in bacteria but can also be found in eukaryotes and archea. (yeast and fungi)
plasmids are used as they can self replicate.

Viruses as vectors
Viruses are commonly used as vectors, In the process of gene therapy. Which is currently used to treat diseases such as cystic fibrosis.
The viruses are genetically modified to carry non-mutated genes into the host cell.
Many different types of viruses can be used (retroviruses, lentiviruses and adeno-associated viruses).
Liposomes as vectors
Liposomes are small spherical vesicles with a phospholipid layer.
These vesicles can also be used in gene therapy to carry non-mutated genes into host cells.
The advantage of using liposomes as a vector is that they can fuse with the cell surface membrane.
genetic fingerprinting how is the pattern of fragments transferred to a nylon membrane
southern blotting
what’s southern blotting
a thin nylon membrane (nitrocellulose) is laid over the gel.
The membrane is covered with several sheets of absorbent paper, which draw up the liquid containing DNA by capillary action
this transfers DNA fragments to the nylon membrane in precisely the same relative location they occupied on the gel.
DNA probes then hybridise to complementary sequences and are visualised but UV light and autoradiography.
