6.3 Manipulating Genomes Flashcards
(24 cards)
Describe the chain termination method of sequencing (Sanger Sequencing)
- Prepare four test tubes with the single stranded template DNA, DNA polymerase, DNA primers, free nucleotides and one of four of the dideoxynucleotides (one type in each test tube)
- Incubate at temperature near 37 C
- The primer anneals to the start of the template and allows DNA polymerase to bind - DNA replication begins - hydrogen bonds form between complementary bases
- At any point, DNA polymerase inserts a dideoxynucleotide = termination of DNA replication = in each test the, the fragments end with the same nucleotide = length of fragment varies as random insertion
- Developing strands separated from template DNA and placed in four wells for each test tube - the separated by length using gel electrophoresis.
- Radioactive tag at the end of each length is read and pieces lined up in order to generate a sequence
What is high-throughput sequencing?
- refers to multiple DNA sequencing technologies
- allows simultaneous sequencing of multiple DNA strands
- They are rapid and produce large datasets very quickly
- Most are automated and next-generation sequencing methods do not include gel electrophoresis
What is bioinformatics?
- the storage and analysis of data from biological studies (DNA/mRNA sequences etc)
- high power computers use
- allows for comparisons between other organisms and can be used as model organisms for humans
Why is gene sequencing useful?
- Evolutionary relationships can be studies by comparing genomes
- Genetic variation = high variation - large number of different base sequences between individuals
- Gene expression
- Epidemiology - diseases sequenced to aid control
- Develop synthetic biology
What is a proteome?
The complete set of proteins expressed by an organism
What is synthetic biology?
- Area of study aims to create new biological parts or redesign existing systems
- Involves large altercations to the genome
What is PCR?
-An in vitro method of DNA amplification
- used to produce large quantities of specific fragments of DNA or RNA from very small quantities (even just one molecule of DNA or RNA).
Why is a buffer solution used in PCR?
to provide optimum pH for he DNA polymerase
Why is Taq Polymerase used?
It comes form thermophilic bacterium = does not denature at high temps - involved in first stage of PCR and optimum temp is high enough to prevent annealing of DNA strands that have not been copied yet
What are the stages of PCR?
- Denaturation - double-stranded DNA is heated to 95 C - breaks hydrogen bonds
- Annealing - temperature is decreased to 50-60 C so that primers can anneal to the ends of the single strands
- Elongation/ Extension - temperature is increased to 72 C for at least a minute - this is optimum temp for Taq polymerase to build complementary strands of DNA to produce new identical strands of DNA
What is gel electrophoresis?
- Molecules are separated according to their size/mass
- +vely charged molecules move towards cathode, -vely charged molecules (e.g DNA as phosphate groups are negative) move towards anode
- Can be used so that DNA can be sequenced/ analysed for genetic profiling
How is DNA prepared for gel electrophoresis?
- DNA is collected
- the sample size is amplified using PCR = more molecules of DNA
- Restriction endonucleases used to cut DNA into fragments - enzymes used cut close to the variable number tandem repeat (VNTR) regions - non coding parts of DNA that vary between people
What is the method for gel electrophoresis?
- Create an agarose gel plate in a tank with wells being cut into the gel at one end
- Submerge gel in an electrolyte solution
- Load the fragments into the wells using a micropipette
- Apply an electrical current to the tank -ve electrode connected at end of the plate with wells as DNA will move towards the +ve electrode
- Smaller/shorter masses move faster/ further
- Fragments not visible so transferred to absorbent paper or nitrocellulose - is then heated to separate two DNA strands - Probes added which after x-ray image or UV-light = pattern of bands which is compared to a control fragment of DNA.
What are probes in gel electrophoresis?
Probes are single stranded DNA sequences that are complementary to the VNTR regions.
- Contain an identifier, either a radioactive label (causes robes to emit radiation that makes the x-ray film go dark) OR a fluorescent stain/dye which shines when exposed to UV light
How is protein separated in gel electrophoresis?
- Different R groups have different charges = buffer added to keep pH constant
- proteins prepared by denaturing (to break disulphide bonds) the manipulating them into rod shapes (which are negatively charged) to allow separation by size
- Can show genotypes of individuals by separating polypeptide chains produced by different alleles.
What are some uses of DNA profiling?
- forensic medicine/ criminal investigations to identify subjects
- identify individuals at risk of developing particular diseases as VNTRs are increased incidence of particular diseases
- determine familial relationships (e.g. paternity tests/ immigration cases)
What is genetic engineering?
- The manipulation of the DNA sequences of an organism
- Possible as the genetic code is universal
- Altered DNA = recombinant DNA can be inserted into the organism and therefore it has been genetically modified
What are the uses of genetic engineering?
- Genetic modification of crops to increase crop yield via resistance or increasing nutritional value
- Modification of livestock to increase productivity or give resistance
- Modification of bacteria to produce medicines
What is the method for genetic engineering?
- Identification of the desired gene
- Isolation of desired gene using restriction enzymes to create sticky enzymes or reverse transcriptase
- Multiplication of DNA fragment using PCR
- Transfer into an organisms using a vector (plasmids, viruses, liposomes) and ligase enzyme. Electroporation (via an electric shock) is used to encourage plasmid uptake
- Identification of new DNA fragment via a marker (specific antibiotic resistant gene marker or fluorescent pigment gene inserted at the same time as desired gene)
What are the uses of genetic engineering?
- GM organisms can be used for research or treatments - cost effective to produce large volumes, fast, reliable supply, identical to desired protein
- Bacteria used to produce human insulin - identical to insulin, reliable supply, fewer ethical/ moral concerns, fewer rejection
-GM plants can be resistant to herbicide/ pests, have enriched vitamins/ nutrients (golden rice to produce vitamin A) - GM livestock can be used for pharming to produce medicines, have larger muscle mass
- GM pathogens used in drug development = adenovirus altered to act as a vector in gene therapy
What are the arguments against GMOs?
- Companies charge (patent their product) farmers more money for GMO seeds - cannot regrow seeds as Gm crops do not breed true
- Lack of long term research on human health
- lack of labelling on food so consumers cant make informed decisions
— Pollen from GM crops contaminates non GM crops - Potential reduction in biodiversity
-potential of superweeds - The antibiotic resistance marker gene could transfer to pathogenic organims v superbug
- ethical concerns of manipulating genomes
What is gene therapy?
Involves using mechanisms to a alter persons genetic material to treat/cure diseases
Can replace/ inactivated a faulty gene and insert a new gene
Most gene therapies are still in clinical trials as hard to find delivery systems for alleles - viruses can be used as vectors (retroviruses)
Not permanent in somatic cells but permanent in germ cells
What are the types of gene therapy?
Two types of somatic gene therapy
- Ex vivo - new gene inserted via a virus into a cell outside the body, blood/ bone marrow cells extracted, edited, grown in a lab and then injected back
- in vivo - new gene inserted via a vector into cells inside the body
Describe the process of ex vivo gene therapy
- Adult stem cells removed
- Virus vector (which cannot reproduce) is altered to contain desired gene
- Patients cells are altered after exposure to virus and then grown in a lab
- Transgenic cells injected into patients body
