Manipulating Genomes Flashcards
What is the polymerase chain reaction used for
Generate many copies of DNA fragments - amplification
In PCR what is often used to cut DNA fragments
Restriction enzymes
What does the reaction mixture for the PCR contain
Original DNA
Primers - small sections of nucleotides
Taq DNA polymerase
And free DNA nucleotides
Describe the steps in the PCR (4)
- Double stranded DNA sample
- Heat to 95 degrees - separate strands
- add primers and reduce temp to 55 degreees (allow hydrogen bonds to form) primers anneal (bind)
- raise temperature to 72 degrees DNA polymerase binds and extends primers using free nucleotides
How do restriction enzymes work
Used to obtain DNA fragments from a genome
Some sections of DNA have palindromic sequences (read the same both ways - forward and backward)
Restriction enzymes recognise specific sequences and cut/ digest at these sites - cuts can leave sticky ends
Different restriction enzymes cut at different sequences as shape of sequence is complimentary to shape of active site
Describe gel electrophoresis
- use of electrical currents to separate DNA/RNA fragments by size
- agarose gel used as it has large pores
- nucleotides are negatively charged so are attracted towards anode
- larger fragments can’t move through agarose as quickly as smaller fragments
How are proteins put through gel electrophoresis
- when are they often used
They have different charges so we mix with a chemical first to denature them - they all now have the same charge
- often used in disease diagnosis
What is the method for DNA sequencing
Chain termination method
Enables us to determine nucleotide sequences on DNA fragments
Why are nucleotides marked with fluorescent dyes
To show what nucleotides are present in a sequence
Mark the end of a fragment - chain terminator
Define Pyrosequencing
a nongel-based DNA sequencing technique that detects inorganic pyrophosphate released during DNA synthesis
What are the chain termination method and pyrosequencing used for
DNA fragments smaller than 1000 base pairs
Why do we have to cut genomes into smaller fragments to be able to sequence
Because the chain termination reaction and pyrosequencing can only be carried out with small fragments - less than 1000 base pairs
What is BAC
Bacterial artificial chromosome (agent)
an engineered DNA molecule used to clone DNA sequences in bacterial cells
What are the uses of sequencing
- allowed for the sequence of amino acids in polypeptide chains to be predicted
- development of synthetic biology - creating biological molecules from scratch
- computational biology ( using a computer to study biology) and bioinformatics ( data store) has also developed
- evolutionary relationships - cytochrome C
- epidemiology ( study of diseases)
What does genetic engineering involve
Modifying the genome of an organism by introducing a gene from another organism
What do we call organisms that have been genetically engineered
Transgenic or transformed
What are the three stages of the genetic engineering process - describe them
- Use restriction enzymes to isolate the desired gene
- Recombinant DNA is made - vector DNA is isolated, and cut open using restriction enzyme in step 1. Using same R.E. Means sticky ends are complimentary. DNA fragment inserted into vector DNA using DNA lignase
- Transforming cells - vector is used to transfer recombinant DNA into bacterial cells (host cells)
Bacteriophage injects DNA into host cell which integrates with bacterial DNA
Plasmid vectors are placed in a mixture with host cells and electric field created. This increases permeability of host plasma membranes and allows plasma to enter - electroporation
Describe how recombinant DNA is made in genetic engineering
Vector DNA is isolated
Vector DNA is cut open using restriction enzyme that was used to obtain original desired gene
As same R.E. Used, means the sticky ends of vector DNA are complimentary to DNA fragment
DNA fragment inserted into vector DNA using DNA lignase (joins sugar phosphate)
Part 3 of genetic engineering process
How are cells transformed
Vector is used to transfer recombinant DNA into bacterial cells (host cells)
Bacteriophage injects DNA into host cell which integrates with bacterial DNA
Plasmid vectors are placed in a mixture with host cells and electric field is created, this increases permeability of host plasma membranes and allows plasmids to enter - electroporation
Why have we genetically modified soybeans
They are an important food source but are susceptible to insect pests
GM soybeans contain a gene that codes for a protein that is toxic to some insects
Gene taken from bacteria BACILLUS THURIUNGIENSIS (B+) and put int plasmid from a second bacteria AGROBACTERIUM TUMEFACIENS
What are the two bacterium used to genetically modify soy beans
1 - from where gene is taken
2 - vector DNA
- Bacillus thuringiensis
- Agrobacterium tumefaciens
What are the ethical issues of genetically modifying crops
Pros and cons
Pros: Less use of chemical pesticides
Increased yield and food
Cons: Monoculture - less biodiversity, vulnerable to disease.
What are the pros and cons of genetically modifying pathogens
Pros: Potential treatments of disease
E.g. tumour cells have receptors for poliovirus - GM the virus to inactivate the disease causing genes
Cons: ethical issues -
Lab troubles and mass outbreak of pathogen
If someone with ill intentions gets hold of this knowledge and biowarfare kicks off
Could the GM version um-GM itself and mss outbreak?
What is pharming
Genetically modifying animals for drugs
What is antithrombin deficiency
Produces too many blood clots
How are goats genetically modified to combat antithrombin deficiency
GM a goat embryo and insert it into a female - all offspring produce milk with antithrombin
Selective breed desired offspring
What are the issues with ownership of GM organisms
Companies will want money for the products
Large companies will have a lot of power
Poorer countries might not be able to afford the medicines and crops
What is gene therapy
Altering alleles inside cells to cure genetic disorders
What are the two ways of carrying out gene therapy
- If the disorder is caused by two mutated recessive alleles, then a working dominant allele can be added
- If it is caused by a mutated dominant allele then it is ‘silenced’ - fragment of DNA is inserted into the middle of the allele so it no longer works
Describe somatic gene therapy
What is the problem with this type
Altering somatic cells (diploid)
Cystic fibrosis - recessive disease
Therefore a functioning dominant allele is inserted into cells lining the lungs
Con: offspring would not inherit new gene as sex cells have not been altered
What is germline gene therapy
Altering sex cells (haploid), zygotes and early embryonic stem cells (<8days)
As a result all cells in organism contain altered DNA so MAY pass this onto offspring
Germline therapy in humans is currently illegal
What are the ethical debates of gene therapy
Pros and cons
Pros:
- prolong life of those suffering with life-threatening genetic disorders
- improve quality of life as it eases symptoms
- germline therapy would allow carriers of genetic disorders to conceive offspring without the disorder
- germline therapy could decrease overall number of individuals suffering from genetic disorders and cancers
Cons:
Technology could be used for other reasons
Expensive - could money be going elsewhere
Potential the therapy could cause more harm than good
What are the disadvantages of gene therapy
- inserted alleles may get overexpressed and result in other problems
- vectors could be recognised as foreign and start an immune response
- an allele could be inserted into the wrong place within the DNA and lead to unknown impacts
- effects may be short-lived in somatic therapy
