Manipulating Genomes Flashcards
What are the 5 components needed in DNA sequencing?
DNA for sequencing
DNA primer
DNA polymerase
Normal bases
Terminator bases (with fluorescent tag)
What does DNA sequencing do?
Determined the precise order of nucleotides within a DNA molecule
What are the stages of DNA sequencing?
Mix the DNA to be sequenced with the dna primer, DNA polymerase and excess normal and terminator bases
Place in a thermal cycle
95 degrees - break hydrogen bonds and separate strands
50 degrees for primers to anneal
60 degrees for DNA polymerase to work best and add nucleotides to complementary bases
Each time terminator base is added, the chain stops producing many different lengths chains
Separated by electrophoresis or capillary sequencing when fluorescent markers on terminators are used to identify the final base on each fragment
Order of bases produced shows sequence of the complementary strand to the dna sample to work out the original from
What are bioinformatics?
Software and computing tools to organise and analyse raw biological data
What is computational biology?
Use of data to build theoretical models of biological systems and predict what could happen under certain conditions
What are 3 things bioinformatics and computational biology contribute into the research of?
Genotype-phenotype relationships
Epidemiology
Evolutionary relationships
What are 3 things gene sequencing has allowed to happen?
Comparison between individuals and between species
Sequences of amino acids in polypeptides to be predicted
Development of synthetic biology
What is the synthetic biology?
The design and construction of novel artificial biological pathways, organisms or devices
What is DNA profiling?
Produce an image of the patterns in the non coding satellite regions of introns of DNA
How is DNA profiling done?
Extract DNA and perform PCR
Digest sample with restriction endonuclease enzymes
Separate strands by electrophoresis
Hybridisation - adding radioactive or fluorescent probes (short complementary sequences to known sequences)
X-ray or apply UV light depending on probe used to see evidence
What is DNA profiling used for?
Forensics
Comparing dna sample found at crime scene to suspects
Analysis of disease risk
Some micro satellite regions are associated with increased risk of disease, specific gene markers can be identified and observed in DNA profiles to assess the risk
What are the 3 stages of PCR
Separate the strands of DNA by heating to 95 degrees which breaks the hydrogen bonds between bases
Annealing primers, cool to 55 degrees so primers anneal to ends of the strands
Heat to 72 degrees optimum temperature for taq polymerase and bases are added to the primer making complementary strand
Repeat
Why is taq polymerase used?
Taq polymerase is a bacterial DNA polymerase and it has much higher optimum temperature that human DNA Polymerase so does not denature when heated during PCR
What are the uses of PCR?
DNA Profiling
What are the three stages of electrophoresis?
Load DNA fragments into the wells of the agarose gel strips which contain a buffer solution to maintain constant pH
Electric current is passed through electrophoresis plate making fragments at cathode end move towards the anode due to the negative phosphate group
The smaller fragments move further and faster as mesh structure of gel resists the movement of the larger fragments when smallest has reached the end the current is switched off
Southern blotting - alkaline buffet solution is added to denature the fragments and separate the strands. Nylon membrane is placed on top the many dry absorbent paper sheets this draws up alkaline solution by capillary action but dna fragments can’t get through so transferred to the membrane the fixed by UV light or 80 degrees
What do you change in electrophoresis when applying it to a protein?
heat it to denture the protein to expose the hydrophobic region/ expose charges
name 4 purposes of a modern botanic garden
conservation of rare endangered species
acts as a gene bank
for teaching/education
as visitor attraction- aesthetic reasons/leisure
what is the role of PCR in sequencing genomes?
to amplify and make many copies of DNA
which will be of different lengths
what is the role of electrophoresis in sequencing genomes?
to put DNA pieces in size order ;
to read, base sequence / order of bases ;
what is the role of restriction enzymes in sequencing genomes?
to cut (genome DNA) into, small(er) / 750 bp, fragments ; to cut, vectors / BACs / plasmids, (for gene library) ;
why does a genome have to be fragmented before sequecning?
genome, too big / very large ;
accuracy better / fewer errors (with small fragments) ;
divide job over, time / different labs ;
what can be used as a vector for bacteria?
plasmid
BAC
bacteriophage
what can be used as a vector for plants?
plasmid
virus
argobacterium tumefaciens
what can be used as a vector animals?
virus
plasmid
BAC
state the 3 main stages of genetic engineering
isolation of the desired gene
inserting gene into a vector
inserting vector into the organism
describe how the gene is isolated in genetic engineering
by restriction endonuclease enzymes
at specific base sequences - palindromic sequences
recognition site is complementary to the enzyme
leaves sticky ends which are unpaired bases at each end of the fragment or leaves blunt ends
describe how vectors are used in genetic engineering
the same restriction endonucleases used to cut the DNA fragment are used to cut the vector - for example a plasmid
this means the sticky ends on the vector are complementary to the DNA fragment containing the gene
the vector DNA and DNA fragment are mixed together with DNA ligase which joins the sugar phosphate backbone.
forms recombinant DNA
what can be done to encourage the cell to take up the vector e.g. the plasmid?
electroporation
bacteria cells are mixed with the plasmid vector and placed in electroporator
an electric feild is applied
this increases permiability of bacterial cell membrane which allows them to take in the plasmid
give an example of genetic manipulation in microorganisms
genetically modified pathogens for research
give an example of genetic manipulation in plants
genetically modified soya for insect resistance
give an example of genetic manipulation in animals
pharming- genetically modified animals to produce pharmocueticals
what is gene therapy?
altering alleles inside cells to cure genetic disorders using vectors such as viruses, plasmids and liposomes
what are the 2 types of gene therapy?
somatic and germ line
what is somatic gene therapy?
altering body cells , particularly those that will be most affected
what is germ line gene therapy?
altering the alleles in germ cells - sex cells or early embryo so every subsiquent cell of organism is affected
what are positive and negatives of somatic gene therapy?
pros
cure / reduce symptoms / better quality of life / less medication;
cystic fibrosis / SCID / Parkinson’s / thalassaemia / LCA ;
extend lifespan / saves lives ;
cons
virus vector may cause (viral) disease ;
procedure may be, invasive / dangerous / painful / stressful ;
temporary / needs to be repeated / limited success ;
immune system / rejection, problems ;
animal testing concerns ;
what are the negative ethical issues of gene therapy?
they could be used in non medical ways- for cosmetic reasons
could do more harm than good - risk of overexpression of the gene
it is expensive so questionable wether this is the best way health services could spend their resources
what are the 5 main stages of genome sequencing?
extract samples of DNA from cells
cut DNA into sections of varying lengths
amplify the DNA
sequence short sections of DNA
place sections in order by matching overlapping regions
what is electrofusion?
another way of producing genetically modified cells
tiny electric currents are applied to the membranes of 2 different cells, this fuses the cell and nuclear membrane of the 2 cells to form a hybrid or polyploid cell containing DNA from both-
used successfully to produce GM plants
how can bioinformatics be useful in determining wether a newly sequenced allele causes genetic disease
base sequence of normal allele and (known) alternatives
held (in database)
computational analysis allows rapid comparison of
sequences with newly sequenced allele
amino acid sequence / protein structures, also held
(in database)
idea of
computer modelling of new protein structure from base
sequence
why are only non coding sections of DNA selected for profiling a human?
in most people, the genome is very similar / most genes the
same
using coding sequences would not provide unique profiles
(parts of) non-coding DNA contains variable numbers of ,
short tandem repeats / repeating sequences
why are proteins heated before being placed in electrophoresis gel?
to denature the protein AND exposes charges or hydrophobic region
