6 - ICH - Genomes & Gene technology Flashcards
What is another name for gene technology? Why?
Recombinant DNA technology
It involves putting the DNA from one organism into another organism
What is a transgenic organism?
Transgenic organism = Organisms that contain DNA from another organism
Summarise the method that is taken to create a transgenic organism? (6)
Isolation
- The DAN sequence making up the required gene is identified
Restriction
- DNA is cut in the correct places to remove the gene
Ligation
- Gene is inserted into a vector, usually a plasmid or virus
Transformation
- Vector carries the gene into the host, usually a bacterium
Selection
- Transformed bacteria that carry the gene are isolated
Culturing
- Bacteria carrying the gene are cultured on a large scale and the product is then harvested
State the 4 different enzymes involved in gene technology?
Restriction endonucleases
Reverse transcriptase
DNA polymerase
DNA ligase
Restriction endonuclease
- What are they
- Role
- Properties (4)
Restriction endonuclease = enzymes used to cut sections out of a DNA molecule
Found in bacteria where they break down the DNA of invading phages (type of virus) ∴ restrict viral replication
- Each restriction enzyme binds to + cuts DNA at a specific target site
- Target site is commonly 4-6 bases long and is symmetrical
- Bacterium’s own DNA is protected from attack by not having the target site, or having it hidden
- 2 strands may be cut to give blunt ends or sticky ends
- BUT sticky ends are prefered
How do bacteria protect their own DNA from being damaged by restriction endonuclease enzymes?
Their own DNA is protected from attack by not having the target site, or having it hidden
Reverse transcriptase:
- What is it
- Role
- Use
Reverse transcriptase = Enzyme used to produce single stranded DNA from mRNA
Some viruses (retroviruses e.g. HIV) contain RNA. When they infect a cell they need to convert their RNA → DNA used by the host cell - use reverse transcriptase for this
DNA polymerase:
- Role
Enzyme attach complementary DNA nucleotides to a single polynucleotide strand to produce a double stranded DNA molecule
DNA ligase:
- Role / use
Enzyme used to join okazaki fragments of DNA together
What is the use of gel electrophoresis?
To seperate DNA fragments according to their length
Describe the procedure on how to carry out electrophoresis to seperate a series of DNA fragments by size (6)
- DNA sample is cut into fragmments by restriction endonucleases
- Fragments put into wells at -ve end of a agarose gel plate using a micropipette. A dye is added to the fragments to make them easier to see
- Buffer added to surface of the gel
- Direct current is applied for a fixed time
- DNA fragments are negative due to the PO4- groups ∴ diffuse towards the +ve end of the gell tray
- Smaller fragments move faster and further than longer fragments
- Pattern of DNA bands is invisible at this time, put under blue or florescent dye which attached to DNA is added
- Compare the results to the fragment pattern of a known length of DNA - ladder
Name 3 processes that electrophoresis is used in?
Genome sequencing
Genetic fingerprinting
Genetic engineering
What is a DNA probe?
What is it used for?
DNA probe = Short single stranded piece of DNA that is labelled using a radioactive or flourescent marker and used to identify a particular DNA base sequence
- Used to identify a gene needed for genetic engineering, to aid genome sequencing and to identify allele associated with genetic disease
What does PCR stand for?
Polymerase chain reaction
What is PCR?
- What is it used for?
- Summarise it in 3 steps
Polymerase chain reaction (PCR) = Used to increase the amount of DNA for analysis in genetic fingerprinting or genetic screening
- Involves repeated replication of DNA in a test tube, doubling the amount of DNA with each replication cycle
3 Stages:
-
Denaturation
- Heat DNA to break H bonds
-
Annealing
- Lower temp and add primers which allow DNA polymerase to attach
-
Elongation
- DNA polymerase extends the primers by attaching compleentary DNA nucleotides
Describe the method of how to carry out PCR (6)
Include any necessary temperatures + enzymes involved
Describe + explain 4 methods that can be taken to isolate a gene
Work backwards from protein:
- Make DNA sequence from knowledge of the amino acid sequence of the protein
- Mix appropriate DNA nucleotides together with DNA polymerase which controls the joining of nucleotides
Use mRNA that codes for the protein:
- Mix mRNA for the protein with DNA nucleotides and reverse transcriptase enzyme and DNA with the desired base sequence will be made
Use DNA probes:
- DNA probe = short single strand of DNA that carries part of a base sequence of a gene you’re looking for
- In suitable conditions, a DNA probe will attach to complementary base sequence in the gene you wish to isolate
- DNA probe is flagged with radioactive or flourescent markers so target gene can be identified
Cutting the gene out of its DNA chain:
- Use a restriction endonuclease enzyme which cuts DNA strands at a specific nucleotide sequence - called the recognition sequence of enzyme
How do you produce insulin through genetic engineering of bacteria?
-
Get the insulin gene
- Extract mRNA from ß cells + incubate them with reverse transcriptase enzyme
- Single stranded complementary DNA (cDNA) is produced, complementary strand forms from free nucleotides
- These double stranded DNA molecules carry the code for insulin
- Guanine bases added on to each end to form sticky ends
-
Put the gene into a vector
- Vector used in this example = plasmid - rings of bacterial DNA, useful as vectors as DNA is frequently swapped in plasmids between bacteria
- Seperate plasmid from the rest of the bacterial cells. Cut pasmid open using restriction endonuclease enzymes. Sticky ends added, this time with cytosine
- Cut plasmid, cut human DNA and DNA ligase is mixed together. C and G bases pair up - forms recombinant DNA
-
Insert recombinant DNA into the host bacterial cells
- Mix plasmids with host bacteria
- NOTE even with encouragement < 1% of bacterial will take up the plasmids containing insulin gene
- Bacteria that’s transformed (picked up insulin gene) will be isolated
- Seperated by antibiotic resistance - tetracyline and ampicillin
- Transgenic bacteria is then cultured
Production fo golden rice:
- What is it
- Purpose
Golden rice = Genetically engineered variety of rice, which the ability to accumulate beta-carotene in its endosperm (food store for seed)
Beta-carotene makes the rice yellow, hence “golden rice”
- Done to help those in poor contries overcome vitamin A deficiency because beta-carotene is converted into vitamin A in the human gut
Benefits and hazards of using GM organisms:
- Pathogens
- Mice
- Golden rice
- Microorganisms
Pathogens:
- +ve = GM virus which have no harmful effect can be used to make vaccines
- +ve = gene therapy
- -ve = In gene therapy, gene modification can increase risk of cancer
- -ve = people worried about being infected with a live pathogen
Mice:
- +ve = GM mice used for medical research
- -ve = welfare concerns
Golden rice:
- +ve = Contains beta-carotene to reduce rates of blindess in children suffering from vitamin A defficiency
- -ve = GM food doesn’t sit well with the general population
Microorganisms:
- +ve = GM E.Coli used to make human insulin to treat diabetics
- -ve = GM microorganisms must be carefully contained. as if they’re released into the wild their genes will spread and affect the ecosystems
What is gene therapy?
Gene therapy = Treatment of a genetic disorder by altering an individual’s genotype
Name the 2 different types of gene therapy
Somatic cell therapy
Germ line therapy
Somatic gene therapy
- What is it?
- Example
Treatment involves altering the alleles in body cells that are most affected by the disorder (somatic cells) i.e. cells not involved in reproduction
Example - CF:
- CF is a recessive genetic disorder that’s very damaging to the respiratory system
- Somatic therapy for CF targets epithelial cells lining the lungs
Germ line therapy
- What is it?
- Example
Treatment involves altering the alleles in the sex cells. This means that every cell of any offspring produced from these cells will be affected by the gene therap and they won’t inherit the disease
- Currently illegal in humans though
Positive ethical issues surrounding gene therapy (4)
- Gene therapy could prolong lives of people with life threatening genetic disorders
- Gene therapy could give people with genetic disorders a better QOL if it helps to ease symptoms
- Germ line therapy would allow the carriers of genetic disorders to concieve a baby without that disorder
- Germ line therapy could decrease the no’ of people that suffer from genetic disorders and cancer
- Beneficial for individuals and society as a whole since fewer people would need treatment
Negative ethical issues surrounding gene therapy (3)
- Technology could potentially be used in ways other than for medical treatment
- E.g. treating the cosmetic effects of ageing
- Potential to do more harm than good by using technology
- E.g. risk of overexpression of genes (too much of a protein)
- Concerns that gene therapy is expensive
- Some believe heath service resources could be better spent on other treatments that have passed clinical trials already
Disadvantages of gene therapy (6)
- Body could identidy vectors as foreign bodies and start an immune response against them
- An allele could be inserted into the wrong place in the DNA
- Possibly cause more problems e.g. cancer
- An inserted allele could get overexpressed (too much of the missing protein) ∴ cause other problems
- Effects of treatment may be short-lived in somatic therapy
- Patients may have to unergo multiple treatments with somatic therapy
- Might be difficult to get the allele into specific body cells
2 disadvantages of somatic gene therapy
- Effects of treatment may be short-lived in somatic therapy
- Patients may have to unergo multiple treatments with somatic therapy
What is DNA fingerprinting?
In what situation is it used?
DNA fingerprinting = using very small samples of DNA to identify individuals by looking at the unique patterns it posses
- Very useful in forensics
What is another name given to polymerase chain reaction?
Genetic profiling
What percentage of our DNA are introns and exons?
Approx 90% = non functioning - intron DNA
Only approx 10% actually codes for things - Exon DNA
What are minisatellites?
Minisatellites = DNA sequences within introns that are repeated
Decribe the sequence of events required for DNA profiling (7)
- DNA is extracted from sample + purified
- DNA is digested into fragments by restriction endonuclease enzymes. An individual’s DNA can be digested to produced a unique mixture of fragments
- Fragments are sorted out by size using electrophoresis
- Current is passed through a agarose gel containing a mixture of DNA fragments. Electrical current is applied. Smaller ones move further/ faster
- DNA fragments are heated to break H bonds to get 2 single strands
-
Southern blotting:
- DNA fragments are transferred to a nylon membrane. At this stage bands are not visible
- Radioactive/ flourescent markers or probs added that bind to the DNA bands
- Nylon membrane is laid on x-ray film which develops and shows the pattern of the DNA bands
Define genome
Genome = All the genetic material of an organism
Why is knowing the sequence of the genome of an organism useful? (2)
- Give info about the evolutionary links between organisms
- Allows the identification and location of particular genes that may affect the risk of development different diseases
Name 2 methods that can be used to sequence a genome?
- Interrupted PCR and electrophoresis but it’s not very efficient
- Main method = chain-termination method
Describe the steps taken to sequence a genome (6)
Chain-termination method:
- Primer binds to the DNA allowing DNA polymerase to attach
- DNA polymerase adds free nucleotides ∴ strand increases in length
- When a nucleotide with a terminator base is added, the DNA polymerase is thrown off ∴ DNA cant get any longer
- Many copies of different lengths of DNA are made, each with a different ‘coloured’ tag at its end
- DNA lengths are seperated by size using electrophoresis
- Laser automatically reads the colour sequence of the tagged nucleotides from the DNA strand with 1 added nucleotide, then 2 added nucleotides, then 3 and 4 etc… until the whole base sequence is determined
