gene technology Flashcards
what is the genome
a genome is a full set of genes in each cell
a genome can be sequenced
what are sequencing projects
the sequencing projects read the genome of a variety of organisms
Determining the genome of a simple organism allows you to know the sequence of bases that codes it
how do we sequence complex organisms
in more complex organisms the presence of introns means that knowledge of the genome cant be translated into the proteome
the proteome is the full range of proteins that can be encoded by the genome
what is the application of sequencing projects
is allows genome-wide comparison between different species allowing the determination of evolutionary relationships
Therefore we can find out about a common ancestor
Genome sequencing is also beneficial to medical research as genome comparison between individuals can allow the development of PERSONALISED MEDICATION
What is the human genome project
it has determined the sequence of bases in a human genome
what is the application of the human genome project
- screening for abnormal/mutated sequences
- allowing identification of disorders before symptoms arise
- Pre-implantation screening which is screening embryos for lethal alleles
- identification of potential antigens to use in vaccines
what are the ethical issues concerning the human genome project
ethical concerns such as the misuse and discrimination of genetic information/ data
what are the different sequencing methods
sequencing methods are constantly developing and are becoming faster and more efficient to use
Sequencing has now become computerised; once the human genome took 15 years to sequence but nowadays to can take up to as little as 26 hours
what is recombinant DNA technology
it involves the transfer of fragments of DNA from one organism (or species) to another
why can transferred DNA fragments be translated within cells of the recipient
the genetic code, transcription and translation machinery are universal, so transferred DNA fragments can be translated within cells of the recipient organisms
what does transgenic mean
it is the word used to refer to the recipient organisms of the foreign DNA by recombinant technology
what are the three different ways that DNA fragments can be formed and isolated
- reverse transcriptase
- restriction endonuclease
- gene machine
what is a reverse transcriptase
reverse transcriptase makes DNA copies from mRNA
They naturally occur in retroviruses
retroviruses are viruses that main genetic information comes from RNA not DNA
how does reverse transcriptase make DNA fragments
- the cell that produces the proteins that scientists want is chosen and should have a large amount of mRNA for the protein
- Reverse transcriptase can align and join the complementary DNA bases to the mRNA bases
- this single-stranded DNA is called complementary DNA (cDNA)
- you need to make the DNA double-stranded and this is done by DNA polymerase
- the cDNA does not have introns
what is a restrictive endonuclease
restrictive endonucleases are enzymes that “cut” DNA at restriction sites to form DNA fragments
as it is an enzyme, the restrictive endonucleases are complementary to specific restriction sites
how do restrictive endonucleases work
some cut through the same location in the double-stranded DNA to produce a blunt end
this means that no bases are exposed or overhang
some creates a staggered ends with exposed bases
These are palindromic and are called “sticky ends” because they can join to complementary DNA bases pairs
what does palindromic mean
the same but backwards
what is the gene machine
it is a computerised way to create DNA fragments which is faster and more efficient
how does the gene machine work
- scientists identify the amino acid sequence of the protein of interest
They then deduce the mRNA sequence from that and the DNA sequence from the mRNA sequence - The DNA sequence is entered into a computer, whciuh has to pass biosafety and biosecurity checks
- the computer creates small sections of overlapping DNA strands called olinguncleotides
- the oligonucleotides can then join to form the DNA sequence of the entire gene
why must we amplify the DNA fragments produced
to use in experiments, these DNA fragments have to be amplified - lots of them have to be made
what are the different ways in which a DNA fragment can be amplified
in vitro cloning: PCR (polymerase chain reactions)
in vivo cloning
how does In vitro: PCR cloning work
- DNA fragments are heated to 95C so hydrogen bonds between base pairs break, producing 2 single DNA strands
- the mixture is then cooled to 55C to allow primers to anneal to the DNA fragments by complementary base pairings
- the mixture is heated to 72C as this is the optimum temperature for DNA polymerase. DNA polymerase attaches nucleotides together to form new double-stranded DNA fragments
what are primers
primers are short sequences of DNA that allows the attachment of DNA polymerase
what does the mixture in In vitro: PCR contain
the mixture in this reaction contains:
-DNA fragments
- DNA polymerase (and helicase)
- primers
- magnesium ions (that act as a co-factor for DNA polymerase)
how does In vivo transformation work
- DNA is cut using restriction endonuclease to create fragments with sticky ended
- A protomer and terminator region are added to allow transcription
- the same restriction endonuclease is used to cut open the plasmid ( which is a DNA loop in bacteria that acts as a vector). This means that the sticky ends of the plasmid DNA is complementary to the sticky ends containing the gene
- the enzyme DNA ligase is used to incorporate the DNA fragments into the plasmid - the recombinant DNA is formed
- the recombinant plasmid is then transferred into bacteria via heat shock or Ca2+ to increase membrane permeability
what are a protomer and terminator
a protomer marks the beginning of the target gene
a terminator marks the end of a target gene
what is a vector
a vector is something used to transfer DNA into cells
why are bacteria used in In vivo cloning
bacteria do not contain introns
why are marker genes important
not all plasmids will take up the foreign DNA fragments and not all recombinant plasmid will be taken up by the bacteria cells
therefore we need to identify which bacterial cells have taken up the recombinant plasmids.
We can do this through marker genes
what are maker gens
these are used to see if plasmids were taken up by the bacteria
how can we find out if recombinant plasmids were taken up by the bacteria
plasmids contain antibiotic resistance genes
we grow bacteria in agar plates containing antibiotics e.g. ampicillin
If the bacteria have taken up the plasmid, they will survive as they will contain antibiotic resistance genes
how do we find out if the plasmids are recombinant
- A DNA fragment is inserted in the middle of a marker gene e.g. GFP, which encodes for fluorescence
- GFP will no longer be made, so the plasmids that do not fluoresce are recombinant and those that do are non-recombinant- they have not taken up the foreign DNA fragments
- this can be done in antibiotic-resistant genes too, recombinant plasmids won’t survive on your plates with antibiotic
what is gene therapy
gene therapy is the mechanism by which genetic disorders can be cured or treated by masking the effect of the faulty allele with the insertion of the functional allele
how does gene therapy work
- the healthy alleles are isolated into cells using vectors
- if a mutant allele is recessive, a dominant allele is inserted and if the mutant allele is dominant, DNA is inserted into the middle of it to silence it
what are the two main types of gene therapy
somatic
germline
what is somatic gene therapy
this is when alleles in body cells are altered (in being that has already matured), therefore changes are not passed on to the offspring
what is germline gene therapy
alleles in sex cells are altered and are passed on to offspring but are considered unethical due to the concern over designer babies or the safety of gene therapy
what are DNA probes
DNA probes are short sections of DNA that are complementary to a known DNA sequence e.g. a mutated allele
They are usually labelled with a fluorescent or radioactive tay
what are DNA probes used for
they are mainly used for genetic screening: which is the study of an individual’s DNA to identify whether they possessed a mutated allele that causes a particular disease
how do DNA probes work
- the labelled DNA probe is mixed with denatured DNA samples from the individual
- if the individual has the mutant allele, the probe will bind to the complementary base sequence in one of the DNA strands - this process is called hybridisation
- this hybridised DNA ( it is the combination of the mutant allele and the probe) us detected using radiation and fluroecence
why is the DNA denatured
it is important that the DNA is denatured so it does not replicate
what is DNA hybridisation
this is when DNA sequences of different species or individuals have complementary base pairs are mixed and therefore hybridize
the more closely related the species are, the higher temp it takes the hydrogen bonds between the two strands.
what are the uses of DNA probes
- genetic screening to see if an individual is a carrier
of a recessive mutation or to evaluate the risk of developing diseases e.g. cancer ( if the disease was dominant, the characteristic would be displayed in the phenotype)
-allows for personalised medicine
what is genetic counselling
individuals may have genetic counselling after the screening, which provides info and support about the results of the screening and how you can lower your risk of getting a particular disease
what is genetic fingerprinting
it is the method used to provide a specific pattern of DNA bands from an individual’s genome
we can look at how closely related individuals are and compare DNA obtained from a crime scene with a suspect
what are VNTRS
genetic fingerprinting uses pieces of DNA called VNTRS (variable number tandem repeats)
they are short sequences of DNA that have repeated themselves in variable numbers in non-coding regions
why are VNTRS useful in genetic fingerprinting
in every individual they vary in length and the number of repeats, so the probability of 2 individuals having the same VNTRS is very low
this is why they are very useful in genetic fingerprinting
how does gentic fingerprinting work
- extraction of the DNA of interest and amplication by PCR
- we need to digest the DNA so we can get the VNTRS that we want. We can do this using specific endonucleases to get the DNA fragments ( the VNTRs)
- The DNA fragments are separated by gel electrophoresis
- The DNA fragment is inserted at the top of the plate in gel electrophoresis. An eclectic current is switched on and the DNA fragments move towards the positive electrode.
This is because DNA fragments are negatively charged (due to the phosphate groups) - the VNTRs are hybridised at specific complementary bases by DNA probes
- the gel is then developed. This means that patterned bands can be visualised by placing the gel on an X-ray film as the probes can emit radiation - this is because the probes often contain radioactive tags
- this reveals the positions of the bands
why do bands move down the electrophoresis gel
the smaller fragments move further down the gel but the larger fragments will be found near the top
The different sized fragments are separated into bands (if a band is, therefore, thicker, there is a higher amount of DNA of that specific size)
what is DNA profiling
we can determine genetic relationships by looking ay how similar the bonding patterns are
what are the uses of DNA profiling
- Forensics
- Medical diagnosis
- plant and animal breeding
