3.8.4 gene technology Flashcards
what is recombinant DNA technology
transfer of DNA fragments from one organism to another
why can the DNA fragment be translated in a different organism
genetic code and translation mechanisms are universal
3 methods of producing DNA fragments
using restriction endonuclease
using mRNA
using a gene machine
describe how DNA fragments are produced using restriction endonuclease
restriction endonuclease cuts DNA at a specific recognition site either side of the desired gene.
forming complementary sticky ends
describe how DNA fragments are produced from mRNA
isolate the mRNA of the desired gene.
reverse transcriptase is used to form single stranded complementary DNA - cDNA
DNA polymerase will be used to join adjacent nucleotides together to create double stranded DNA, using cDNA as a template
why is using mRNA better than using DNA to obtain DNA fragments
there will be more mRNA of the desired gene, easily extracted
in mRNA the introns are already removed via splicing, so can be inserted into prokaryotes who cant undergo splicing
describe how DNA fragments are produced using a gene machine
synthesises DNA fragments quickly from scratch without using DNA as a template.
the amino acid sequence is entered into a computer, allowing a base sequence to be established.
short sections of DNA are made, around 20 nucleotides long (oligonucleotides)
the oligonucleotides then join together to make the required gene
what are in vivo and in vitro techniques used to amplify DNA fragments
in vivo - culturing transformed host cells
in vitro - polymerase chain reaction (PCR)
explain how DNA fragments are amplified by PCR
DNA fragments, DNA polymerase, primers and DNA nucleotides are used for the reaction mixture.
mixture is heated to 95 degrees to break hydrogen bonds between the strands
the mixture is cooled to 55 allowing primers to bind to the DNA fragment strand
mixture is heated to 72 allowing nucleotides to align across complementary base pair, adjacent nucleotides will be joined via DNA polymerase forming phosphodiester bonds
the cycle is repeated, where amount of DNA doubles
explain the graph of a PCR
shallow gradient at start as there is small number of DNA during first divisions
steep gradient due to exponential growth
shallow gradient at end as graph plateaus, as there are no DNA nucleotides left to react.
what do primers do in PCR
primers are short single stranded DNA fragments, complementary to the DNA base sequence at the start of the desired gene, allowing DNA polymerase to bind
2 primers are needed at each 5’ end
explain how DNA fragments are amplified in vivo
promoter and terminator regions are added to DNA fragments
DNA fragments and gene markers are inserted into vectors using restriction endonuclease and ligase
identify the host cells which have took up the DNA fragment by using the gene marker
why are promoter and terminator regions added to DNA fragments
promoter - allows transcription to start by allowing RNA polymerase to bind to the DNA
terminator - ensures transcription stops at the end of the gene by stopping RNA polymerase
explain the role of enzymes in forming recombinant DNA
restriction endonuclease - cut vector and gene to form complementary sticky ends
ligase - join DNA fragment to vector forming phosphodiester bonds between adjacent nucleotides
why are gene markers used
allows detection of GMO
e.g. using antibiotic resistance or fluorescent proteins
this is needed as not all cells are transformed
how is recombinant DNA technology useful
can produce human proteins, more ethically acceptable compared to using animals.
make GM crops resistant to herbicides.
describe gene therapy
introduction of new DNA into cells containing healthy alleles to overcome faulty alleles in people with genetic disorders.
what are issues related to gene therapy
the modified cells have a short lifespan so will need constant treatment.
may trigger immune response against GM cells.
the long term effect is not known.
what are DNA probes
short single stranded DNA with a complementary sequence to a target allele due to complementary base sequencing
what is DNA hybridisation
binding of a single stranded DNA probe to a complementary single strand of DNA, forming hydrogen bonds between the bases
explain the process of identifying a location of specific alleles
extract DNA and amplify using PCR.
cut the DNA at specific base sequences using restriction endonuclease.
separate DNA fragment according to length using gel electrophoresis.
transfer to nylon membrane and treat with alkali to form single stranded DNA.
add a DNA probe which will bind target alleles.
to show specific allele, expose DNA to UV light causing fragment to light up is fluorescently labelled or use autoradiograph if radioactive probe was used.
what is gel electrophoresis
a method used to separate DNA or proteins according length or mass and charge.
how is gel electrophoresis used to separate DNA fragments
add DNA sample into well with gel and cover with buffer.
an electrical current is passed through - as DNA is negatively charged so moves towards the positive electrode
shorter DNA fragments travel faster so travel further.
how is data from gel electrophoresis interpreted
use a standard with known DNA fragments and known lengths.
compare to position of the unknown DNA fragments
when would you use DNA probes
scanning patients for heritable conditions or health risks
what is genetic counselling
discussing treatment and lifestyle changes to reduce risk of genetic conditions
what is personalised medicine
medicine tailored to an individuals genotype, increasing effectiveness of treatment
what are variable number of tandem repeats (VNTRs)
repeating sequences of nucleotides found within non-coding sections of DNA throughout the genome
why are VNTRs useful in genetic fingerprinting
the probability of 2 individuals having the same VNTR is very low, so will be unique to each person
explain how genetic fingerprinting can be used to analyse DNA fragments
extract DNA from sample and amplify using PCR.
cut DNA at specific base sequences using restriction enzymes.
separate VNTRs using gel electrophoresis based on length. transfer to nylon membrane and treat with alkali to form single stranded DNA. add labelled DNA probe which will bind to complementary VNTR.
if fluorescent labelling used, use UV light, if radioactive, use autoradiograph
what are the uses of genetic fingerprinting
compare genetic fingerprints of suspects to genetic fingerprinting of DNA at crime scene.
some VNTR patterns are associated with genetic diseases
showing if organisms are closely related to avoid inbreeding
