Chapter 19- Genetic Technology Flashcards
Define genetic engineering
- Any procedure in which the genetic information in an organism is changed by:
-altering the base sequence of a gene
-introducing a gene from another organism - The organism is said to be a generically modified organism
Define recombinant DNA
This is DNA made by artificially joining together pieces of DNA from 2 or more different species or the same.
Define transgenic organism
An organism that contains DNA from another source, such as from another individual of the same species or from a different species
- This is specific to interspecies gene transfer
Define GMO
Any organism that has had its DNA changed in a way that does not occur naturally or by selective breeding
- This is open to all genetic modifications
Genetic engineering Principles
State the essential steps in which a GMO may be produced:
1- The gene that is required is identified and may be cut from a chromosome
2- Multiple copies of the gene are made using a technique called the polymerase chain reaction
3- The gene is inserted into a vector which delivers the gene to the cells of the organism.
4- The vector takes the gene into the cells
5- The cells that have the new gene are identified and cloned
Define restriction enzymes
This is an enzyme, originally derived from bacteria, that cuts DNA molecules; each type of restriction enzyme cuts only at a particular sequence of bases to give blunt ends or sticky ends
What are the restriction sites
- these are target sites that have specific sequences of 4 to 6 bases.
- Restriction enzymes either cut straight across the sugar-phosphate backbone to give blunt ends or they cut in a staggered fashion to give stick ends
Sticky ends: Short lengths of unpaired bases
How do you find the specific piece of DNA using gene probes
- Gene probes is a single-stranded DNA with a known base sequence.
- It binds with lengths of DNA which have a complementary base sequence
Describe reverse transcriptase
- This is an enzyme found in viruses that have RNA as their genetic material
- This enzyme uses single-stranded mRNA as a template and free DNA nucleotides to form a stranded DNA. This Is with the help of a DNA primer
- The process is complete when DNA polymerase synthesises another polynucleotide to form double-stranded complementary DNA (cDNA)
- The mRNA template is digested by RNA-ase enzyme.
- Sticky ends will then be added for insertion of cDNA into the plasmid
Describe how synthesis of DNA can take place with a DNA synthesiser machines
- First, choose the codons for the amino acid sequence they need.
- The sequence of nucleotides is held in a computer that directs the synthesis of short fragments of DNA in DNA synthesiser machines
- These fragments are then joined together to make a longer sequence of nucleotides that can be inserted into plasmids for genetic engineering.
Define vectors
These transport something from one place to another
Describe how plasmids are obtained
- They treat bacteria with enzymes that break down their cell walls
- They then spin the bacteria at high speed in a centrifuge so that the larger circular DNA is separated from the much smaller plasmids
- The circular DNA of the plasmid is cut open using a restriction enzyme. The same enzyme is used to cut the gene that will be used
State the advantages of using plasmids as vectors
- Their small size makes it easy to isolate and modify
- They can replicate independently and rapidly
- They can be used in many types of host cells
- Plasmids are stable in host cells
- They have restriction sites, so can use restriction enzymes
- They have marker genes, so transformed bacteria can be recognized.
Which enzyme links together the sugar-phosphate backbones of the DNA and the plasmids and how do they do it
DNA ligase!!!
It does this by catalysing the formation of phosphodiester bonds, forming a closed circle of double-stranded DNA containing the new gene.
Other than plasmids, name 2 more vectors
- Viruses
- Liposomes
Describe the steps to getting the plasmids into the bacteria (Transformation)
- First, the bacteria and plasmids are put into a solution with a high concentration of calcium ions
- The mixture is cooled and given a heat shock
- This will increase the chances of plasmids passing through the cell surface membrane of the bacteria. A small proportion of the bacteria (1%) take up plasmids with the gene. They are said to be transformed.
Describe one way to produce multiple copies of a gene using bacteria
- DNA polymerase in bacteria will copy the plasmids
- Bacteria will divide by binary fission so that each daughter cell has several copies of the plasmid (Gene cloning)
- The bacteria may also transcribe the new gene and use the mRNA in translation to form a protein. This is called a recombinant protein
Describe how Recombinant human insulin is made step by step
- First the gene is cut out from mRNA for insulin from pancreatic B-cells.
- The mRNA is used as a template for reverse transcriptase to make single-stranded DNA
- The single stranded DNA molecules were then used as the template for DNA polymerase to make double-stranded DNA
- It will carry the insulin genes and will be inserted into the plasmids. The plasmids will then transform the bacterium E.coli
Advantages of Genetic engineering insulin
+There is now a reliable supply available to meet the increasing demand.
+The insulin is human insulin not animal insulin
+Can modify the insuline gene for example making them act faster and
+Cheapter to produce in large volumes
+Fewer ethical, moral, or religious concerns
Describe an example of genetic markers with fluorescent substances
- Enzymes obtained from jellyfish make a protein called GFP which fluoresces bright green ultraviolet light.
- The gene for the enzyme is inserted into the plasmids
- Now all that needs to be done is to identify the bacteria that have taken the plasmid by observing whether they shine ultraviolet light onto them.
- The ones that glow green are genetically modified.
Advantage of using fluorescent genes as markers
- They are easier to identify
- More economical - does not invlove growth of bacteria
- No risk of antibiotic resistance being passed onto other bacteria
Describe the promoter
This is the region of DNA to which RNA polymerase binds as it starts transcription.-
- The promoter can be said to control the expression of a gene and ensure high level of gene expression
- The promoter allows RNA polymerase to bind to DNA and ensures that it recognises which of the 2 DNA strands is the template strand
Define Gene editing
A form of genetic engineering in which the genome of an organism can be changed by deleting, inserting or replacing a length of DNA
What is ‘Crispr’
This is a group of base sequences that code for short lengths of RNA that direct a nuclease enzyme known as Cas9 towards specific sequences.
What is the role of Crispr-associated (Cas9) enzyme
This is an endonuclease that cuts DNA at a point that is determined by these RNA molecules, known as guide RNA(gRNA)
- Part of gRNA has a sequence of 20 bases that can locate and bind to a strand of DNA with the complementary base sequence. The enzyme Cas9 has 2 active sites to cut DNA across both sides.
- This gives engineers a way to edit DNA at specific places within an organisms genome.
What is the everyday use of Crispr/cas9
- Can be used to prepare customised drugs to treat cancers
- Can be used to edit the genomes of insect vectors to prevent the transmission of diseases such as malaria and viruses
- Can be used to target multiple genes at the same time
Define the polymerase chain reaction
a laboratory technique that amplifies a specific DNA segment from a small sample
List the following substances that are added to each tube in the PCR machine
- A sample of DNA
- 2 different short lengths of single-stranded DNA to act as primers for the DNA polymerase
- free dNTPs
- A buffer solution
- A solution of heat-stable DNA polymerase
STEP 1
DENATURATION:
- The DNA is denatured by heating it to about 95 degrees. This breaks the hydrogen bonds between the base pairs and separates the double-stranded DNA, leaving bases exposed
step 2
ANNEALING
- Primer binds to base sequences on either side of DNA (hydrogen bonding)
- The primers are often 20 base pairs long and have complementary bases on either side part of the lenth of DNA
- The primers have different base sequences where on binds to the ‘upward’ strand and the other binds to the ‘downward’ strand
- This temperature is about 60 degrees
step 3
EXTENSION:
- The enzyme DNA polymerase uses dNTPs to build new strands of DNA
- The synthesis of new DNA needs 72 degrees
- This is Taq polymerase which was the first heat-stable DNA polymerase and is found in hot springs
What are the 2 advantages of Taq polymerase
- Does not get destroyed by the denaturation stage so does not need to be replaced
- It has a high optimum temperature. The temperature in the annaeling process therefore foes not have to be dropped .
Define gel electrophoresis
This refers to the seperation of charged molecules such as DNA by differential movement through a gel in an electric field.
Describe the most important factors that affect the movement of charged molecules within the gel
- net charge : Negatively charged molecules such as DNA will move towards the anode(+) and away from the cathode(-). High charged molecules move faster than less charged ones
- Size: The smaller the molecules are, the faster they move through the gel
- Composition of the gel:The size of the pores within the gel will determine the speed
State the two types of gel and what they are used for
Polyacrylamide gel:
- This is used for seprating small fragments of DNA
Agarose gel:
- This is used for seprating fragments that are between 100 base pairs and 50,000 base pairs
Step by step, state the procedure of gel electrophoresis
1.The prep of the gel involves dissolving a powder in hot water. Still fluid is poured into an electrophoresis tank. A ‘comb’ is placed at one end and this results to wells being formed once the gel has solidified
2.When the gel is set, a buffer solution is poured into the tank until it covers the gel in order to give a constant pH
3.A micropipette is used to transfer samples of DNA to all of the wells. The samples of DNA contain a tracking dye in order to track how far they have travelled across the gel
4.A reference sample known as a DNA ‘ladder’ is used to determine the lengths of the fragments of DNA and this can be placed on either ends.
5.A battery pack is connected to the electrodes where the negative electrode is at the same end as the wells, hence allowing the fragments to repel and pull towards the positive electrode.
6.The tracking dye shows how far material in the samples has moved across the gel. when the dye has moved across most of the gel, the battery must be disconnected
7.A stain is added to the gel and is rinsed away to reveal bands across the gel, showing positions of the DNA fragments.
Define Microarrays
These are used to identify the genes present in an organisms genome and to find out which genes are expressed within cells as well as compare the genes present in 2 different species
Describe how microarrays analyse genomic DNA
- DNA is collected from the species that are going to be compared
- Restriction enzymes are used to cut the DNA into fragments
- The fragments are denatured to form single -stranded DNA
- DNA is labelled with specific fluorescent tags(Different colours)
- The labelled DNA samples are mixed together and allowed to hybridise with the probes on the microarray.
- The microarray is inspected using UV light, causing the tags to fluoresce
- The presence of colour indicates that hybridisation has taken place. The complementary bases of the probes have paired with the DNA
- Any DNA that has not bound to the probes will be washed off
- The microarray is scanned so data is read by the computer and stored.
How can microarrays detect which genes are active through mRNA
- mRNA is collected from 2 types of cells and reverse transcriptae is used to conver mRNA to cDNA
- PCR may be used to increase quantity of cDNA as mRNA quantity is too low
- cDNA is labelled with fluorescent tags and denatures to give single stranded DNA
- Single stranded DNA hybridises with probes on microarray
- UV light is shone
Describe Bioinformatics
- They refer to the collection and processing of biological info and data using computer software
Why are most recombinant human proteins produced using eukaryotic cells
- These cells will carry out post-translational modification
- They can produce a suitable human protein
How does recombinant factor VIII treat heamophilliacs
- F8 is inserted into hamster kidney and ovary cells which are then cultured in fermenters.
- The cells constantly produce factor VIII and is extracted and purified before treating heamophiliacs
Describe Severe combined immunodeficiency (SCID) and the enzyme used to treat it
- This is a disease due to the mutation of the gene ADA on chromosome 20 leading to a defeciency of adenosine deaminase (ADA). This enzyme catalyses the break down of deoxyadenosine which is toxic to T-lymphocytes
- Suffereres may die at infancy due to normal infections
- T lymphocytes of patients are removed and normal alleles of the ADA gene are introduced using a virus vector
Describe genetic screening for the faulty alleles of BRCA1 and BRCA2
- These genes produce tumour suppressor proteins and control cell growth
- Faulty alleles of these genes are screened by screening the regions of chromosome 7and 13 where BRCA is located
state the advantages of genetic screening for women
- Can determine whether they are carriers or not
- Can help them make decisions about future pregnancies
- Helps the doctors take special precautions during pregnancy
- Women can use pre-implantation genetic diagnosis (PGD) during IVF to choose an embryo that is carrying the allele for the relevant blood clotting
State the advantages of genetic screening for cystic fibrosis
- It improves prompt treatment
- improves quality of life by early detection
- better future planning
Describe Gene therapy
This involves the insertion of ‘normal’ alleles of a gene into the cells of a person with a genetic disorder in an attempt to cure the disorder
Most common vectors are:
- Viruses
- Liposomes
- Naked DNA
State the social and ethical considerations of using gene therapy
- Potential side effects could cause death
- It is considered a long-term treatment
- The expense of treatments and this makes the therapy accessible to a limited number of people
- There are some cultural/ religious beliefs such as jehova witnesses
- Gene therapies are temporary as cells that are genetically modified have a short life span
- No need for regular injections/treatments
- It is not possible to affect dominant alleles such as Huntingtons disease
What is the need for GM plants
- Weeds would otherwise compete with the crop space, light, water and nutrients from the soil.
- Reduces some costs for farmers and increases crop yield
How do herbicide resistant crops cause negative effects to the environment
- GM plants are more likely to become an argicultural weed
- Pollen will transfer the resistance gene to wild relatives of the crops, producing hybrids which could be invasive weeds
- Herbicide resistant weeds could evolve and become tolerable to the same herbicide
- modified plants may be a direct hazard to humans, domestic animals or other beneficial animals
Describe Bt toxin as an example of insect resistant crops
- Bt toxin is lethal to insects.
- A gene for this toxin has been taken from a bacterium and different strains of this bacterium can be used against different insect species,
- Crop plants that contain this toxin gene produce their own insecticides
How do insect-resistant crops negatively affect the environment
- Evolution of resistance by the insect pests
- can be a damaging effect on other species of insects
Describe the genetically modified Atlantic salmon
- A growth hormone regulating gene from a pacific chinook salmon and a promoter from another species were injected into a fertilised egg
- By producing growth hormone throughout the year, the modified salmon are able to grow all year instead of just in spring and summer.
State the ethical and social implications of using GMO seeds
- GM seeds are expensive. Growers mostly need to buy seeds each season and this will raise costs
- There is danger of losing traditional varieties. This is bad as some of these varieities are important for desirable background genes in some regions where the climate is changing
