6.1.3 - manipulating genomes Flashcards
genome
all the genetic material it contains
introns
the non coding regions of DNA
satellite DNA
short sequences of DNA that are repeated many times
minisatellite
sequence of 20-50 base pairs that will be repeated from 50 to several hundred times
VNTRs
variable number tandem repeats (minisatellites)
DNA profiling
technique to produce an image of the patterns of DNA of an individual
5 stages of producing a DNA profile
- extracting the DNA
- digesting the sample
- separating the DNA fragments
- hybridisation
- seeing the evidence
stage 1 of DNA profiling- extracting the DNA
DNA must be extracted from a DNA sample, a technique called PCRs are used to give scientists enough DNA from a tiny tissue sample
stage 2 of DNA profiling - digesting the sample
the strands are cut into small fragments using restriction endonucleases which cut DNA at restriction sites
stage 3 of DNA profiling - separating the sample
the cut fragments need to be separated using eletrophoresis where the shorter fragments move towards the bottom of the gel and longer fragments remain at the top
they are then transferred from the gel into nylon membrane in a process known as southern blotting
stage 4 in DNA profiling - hybridisation
DNA probes (radioactive or flourescent) bind to complimentary strands of DNA - identify the regions that are more varied
stage 5 in DNA profiling - seeing the evidence
radioactive - x ray images taken
fluorescent - UV light so the tage slow
PCR
polymerase chain reaction - allows scientists to produce a lot of DNA from the tiniest original sample
PCR process
step one - separating the strands by increasing the temperature and breaking the hydrogen bonds
step two - annealing of the primers by decreasing the temperature and primers bind to end of DNA strands
step three - synthesis of DNA where temperature is increased to optimum temperature for DNA polymerase to work, to replicate the DNA strands.
uses of DNA profiling
- forensic science (traces of DNA left at crime scene)
- prove paternity of a child
- identifying individuals who are at risk of developing particular diseases
DNA sequencing
process of determining the precise order of nucleotides within a DNA molecule
who developed the first DNA sequencing process?
Fredrick Sanger
Human Genome Project
international project in which scientists from a number of countries worked to map the entire human genome.
what are terminator bases?
modified versions of the four nucleotide bases. (a,t,c,g), that stops DNA synthesis when they are included (e.g a A terminator base will stop DNA synthesis at a location that an A base would go.
These are given coloured flourescent tag
stages of DNA sequencing
- Dna sequence mixed with primer, DNA polymerase and normal nucleotides
- PCR steps 1 and 2
- at 60C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to DNA template strand
- terminator bases stop the synthesis of DNA, resulting in different fragment lengths
- eletropheresis - flourescent markers used to identify the final base on each fragment, which can then show the sequence of DNA
next generation sequencing
DNA sequencing technologies have led to new automated high thoroughput sequencing processes. All the clusters can be sequenced an imaged at the same time.
bioinformatics
development of software and computing tools needed to organise and analyse raw biological data
computational biology
uses data from bioinformatics to then predict what will happen in different circumstances
significance of analysing genomes of pathogens
- doctors to find out source of an infection
- doctors to identify antibiotic resistant strains of bacteria
- to track the progress of an outbreak
DNA barcoding
identifying particular sections of the genome that are common to all species but vary between them and compare them.
e.g cytochrome c ocxidase
proteomics
study and amino acid sequencing of an organisms enture protein complement
genetic engineering
the manipulation of a genome
basic principles of genetic engineering
involves isolating a gene for a desirable characteristic in one organism and placing it into another organism using a suitable vector.
transgenic
an organism that carries a gene from another organism
first stage of genetic engineering
isolating the desired gene
techniques to isolate the desired gene
- restriction endonucleases to cut the required gene from the DNA of the organism
- isolating mRNA and then using reverse transcriptase to turn it into a DNA strand (cDNA)
sticky ends
regions with unpaired exposed bases after restriction endonucleases have cut it.
easier to insert desired genes into DNA of different organism
formation of recombinant DNA
the DNA is isolated and now must be inserted into vectors that can carry it into the host cell
most commonly used vectors
bacterial plasmids - small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.
recombinant DNA
plasmid combining with the host DNA
marker gene
in plasmids - enables scientists yo determine that the bacteria have taken up the plasmid.
how is the DNA fragment inserted into plasmid
cut open, use a restriction endonuclease, resulting in the plasmid having the same sticky ends as the DNA.
DNA ligase forms phosphodiester bonds, joining them together.
transferring the vector process
culture the bacterial cells and plasmids in calcium rich solution and increase temperature - causes bacterial membrane to become permeable and plasmids can enter
electroporation
electroporation
small electrical current is applied to the bacteria, making the membranes porous and plasmids move into cells
electrofusion
tiny electric currents are applied to membranes of two different cells. this fuses the cell and nuclear membranes to form a hybrid or polyploid cell.
genertically modifying plants example
agrobacterium tumefaciens - a bacteria that causes tumors in healthy plants.
can make crops resistant herbicides etc.
trasgenic plant cells form a callus which can be grown into a new transgenic plant
somatic cell gene therapy
involves replacing the mutant allele with a healthy allele in the affected somatic body cells
vectors used in gene therapy
in vivo - plasmids
in vitro - viruses
why is somatic cell gene therapy only temporary?
somatic cells have limited life and are replaced from stem cells, which will have the faulty allele
germ line cell gene therapy
insert a healthy allele into the germ cells - usually eggs - and this will b passed down to own offspring
why is germ line gene therapy illegal in this country?
human rights violation - no consent
impact on whole organism
designer babies
how can scientists use a gene to study relationships between organisms
sequence the gene for different organisms
compare the base sequences - those that are more similar are more likely to be closely related
