gene technology Flashcards
what is recombinant DNA technology?
transfer of fragments of DNA from one organism to another
DNA that contains nucleotides from other organisms
3 ways of producing DNA fragments
- reverse transcriptase
- restriction endonucleases
- gene machine
how can reverse transcriptase make DNA fragments?
- remove mRNA from a cell
- add reverse transcriptase and DNA nucleotides
- converts the mRNA into DNA (double stranded)
uses mRNA as template
= cDNA (complementary)
why is mRNA used for reverse transcriptase?
easier to isolate from cells than DNA
introns already removed in splicing
cDNA produced is complementary to mRNA, therefore is a copy of original DNA
how can restriction endonucleases make DNA fragments?
- obtain sample of DNA from a cell
- incubated with specific restriction endonuclease
- cuts DNA fragment using hydrolysis
how do restriction endonucleases work?
- different endonucleases cut at different base sequences
- shape of sequence is complementary to enzymes active site
- used to obtain gene between the sequences to cut it out, hydrolysis
(gene cant contain specific sequence or it will be cut)
leaves sticky or blunt ends
what are sticky and blunt ends?
when a gene is cut using a restriction endonuclease, leaves:
sticky ends - one strand of fragment longer than the other
- easier to insert gene as hydrogen bonds can form between bases
blunt ends - when both strands are same length
- nucleotides can be added to create sticky ends
how does a gene machine produce DNA fragments?
- determine amino acid sequence of the protein
- first nucleotide fixed to support
- nucleotides added step by step in order
- creates oligonucleotides (short sections of DNA)
- oligonucleotides joined to make longer fragments
ways of amplifying DNA fragments
PCR (in vitro)
in vivo
describe PCR as a method to amplify DNA fragments
- reaction mixture made
- heated to 95c
- hydrogen bonds break, strands separate - cooled to 55c
- allows primers to bind - heated to 72c
- allows complementary nucleotides to attach (optimum) - heat stable (taq) DNA polymerase joins nucleotides together
- with phosphodiester bonds
repeats
- each PCR cycle doubles amount of DNA
what does the reaction mixture for PCR contain?
- DNA sample
- free DNA nucleotides
- primers
- DNA polymerase (taq)
in vivo method of amplifying DNA fragments
- use restriction endonuclease to obtain desired gene from DNA
- use same RE to cut open a plasmid
- leaves sticky ends complementary to sticky ends of fragment - join desired gene to plasmid using DNA ligase
- joins sticky ends
creates recombinant DNA (vector and fragment)
- vector (plasmid) inserted into a bacteria cell
- plasmid replicated during binary fission to create more recombinant DNA (and therefore gene)
what are transformed cells and how can you identify them?
cells that have taken up the vectors, contain plasmids with recombinant DNA
- marker genes
how do marker genes identify transformed cells?
used to identify which cells contain the plasmid with the desired gene
- another gene (for an identifiable characteristic) is inserted with the desired gene
- means transformed cells also contain that gene
- eg antibiotic resistance, so only transformed and resistant cells survive
or gene inserted within a gene for antibiotic resistance
tranformed are killed
benefits of recombinant DNA tech in agriculture
crops can be transformed to be:
- more nutritious
- higher yields
reduce risk of famine
- pest resistant (fewer pesticides needed, cheaper)
benefits of recombinant DNA tech in industry
industrial process often use enzymes
- can be made from transformed organisms
- made in large quantities and cheaper
benefits of recombinant DNA tech in medicine
transformed organisms used to make drugs and vaccines
- can be made cheaper and quicker
eg insulin
issues of recombinant DNA tech in agriculture
monoculture of transformed crops - genetically identical
- makes whole crop vulnerable to same disease - famine risk
- reduces biodiversity
long term effects of GM food on health unknown
issues of recombinant DNA tech in industry
some consumer markets won’t import genetically modified food - producers suffer economically
no labelling - no choice over eating GM food or not, unethical
issues of recombinant DNA tech in medicine
could lead to unethical uses
eg designer babies - modifying and choosing characteristics
can be used to save lives but companies may limit use for economic reasons
humanitarian benefits of recombinant DNA tech
- agricultural crops made that can reduce risk of famine and malnutrition
eg drought resistant crops - used to make vaccines and drugs available to more people
eg made chpear so more people can afford them - can be used in gene therapy to treat diseases
environmentalists issues with recombinant DNA tech
monocultures (of transformed crops crops) reduce biodiversity
anti-globalisation activists issues with recombinant DNA tech
oppose globalisation (growth of large multinational companies)
few large companies control forms of genetic engineering
as use increases, these companies get bigger and more powerful
- smaller companies unable to compete
what does gene therapy do?
used to treat genetic disorders
it alters the defective genes (mutated allele causing disease)
done by inserting DNA fragment into persons original DNA
how does gene therapy work to change the mutated gene?
if the mutated alleles are:
2 recessive - add dominant to supplement them
1 dominant - silence it (add DNA to middle of allele so it doesn’t work anymore)
2 types of gene therapy
somatic - altering alles in body cells
particularly those affected by disease
- eg epithelial cells in lungs for cystic fibrosis
(doesn’t affect sex cells, could be inherited)
germ line - altering alleles in sex cells
disease can’t be inherited as every sex cell has been altered
how is gene therapy done?
working allele inserted into cells using vectors
vectors include:
- altered viruses
- plasmids
- liposomes (spheres of lipids)
what is a DNA probe?
short strands of DNA
have a specific base sequence complementary to the target allele
means they will bind (hybridise) to the target allele if present
has a label attached so it can be detected
- fluorescence (use UV light)
- radioactive (use x ray film)
how does a DNA probe work?
DNA fragment obtained
(restriction endonucleases and separated using electrophoresis)
- transferred onto a nylon membrane
sample of DNA incubated with DNA probe (with label)
if target allele present, DNA probe will bind to it
- probe not removed when washed
use UV light or x-ray film to detect DNA probe, if present
DNA in sample must be single stranded to allow the probe to bind
what is a DNA probe used for?
locating specific alleles of genes
see if persons DNA contains mutated alleles that causes genetic disorders
used for screening
uses of screening with DNA probes
- identify heritable conditions
screened to identify mutated allele
- means treatment can be started earlier eg in cystic fibrosis - determine how patients will respond to drugs
screening for a specific mutated allele can show if someone has it so if a specific drugs will work - identify health risks
inheriting a particular mutated allele can increase risk of developing certain cancers
- allow them to make life choices to reduce their risk
however could lead to discrimination by insurance companies or employers if some people have a higher risk of disease
uses of DNA probes (3)
locate specific alleles
can screen for:
- heritable conditions
- responses to drugs
- health risks
what is genetic counselling?
advising patients and families about the risk of genetic disorders
advise on screening and the results
- the risks of it being passed on to offspring
- advice on future steps eg most effective prevention or treatments
- explain potential consequences of disease eg economically
screening can help identify if someone is a carrier of a mutated allele and what type (showing what genetic disorder it causes)
what is personalised medicine?
medicines that are tailored to an individuals DNA
genes mean different people respond to drugs in different ways - makes them more effective for some than others
can be used in prevention if mutated allele detected
if doctors have genetic information it can be used to predict responses to drugs
therefore prescribe the most effective
benefits of genetic screening
can identify people with allele who are at risk of developing specific diseases
- can make lifestyle choices to reduce their risk eg diet
can help potential parents chose to have children or not
- may not want to risk passing on harmful alleles
limitations of genetic screening
designer babies - unethical
could lead to discrimination in workplaces or for insurance
- for people who have a higher risk of developing certain diseases
why are peoples genetic fingerprints different?
genome contains variable number tandem repeats
- base sequences that don’t code for proteins and repeat next to each other
number of times the sequences are repeated = length in nucleotides
- this varies between people
repeats also occur at different places in the genome
- varies between people
probability of 2 people having same genetic fingerprint is very low
- need same number of VNTRs at same place in genome
how are the fragments obtained for electrophoresis?
restriction endonucleases used to at specific base sequences to cut VNTRs out
results in fragments of varying lengths - depending on number of repeats
apified using PCR = many copies
why are the fragments negatively charged?
DNA contains a phosphate group
= negative
so attracted to positive electrode at end of gel
describe electrophoresis (how a genetic fingerprint is made)
sample of DNA obtained (blood, saliva etc)
PCR used to make copies of area of DNA fragment containing VNTRs
electrophoresis used to separate them
fluorescent tag added - visible in UV
- DNA mixture placed in well in gel
covered in a buffer solution that conducts electricity - electric current passed through gel
DNA fragments negatively charged
move towards positive electrode at far end of gel - small DNA fragments move faster so travel further
larger ones don’t travel as far
- DNA fragments separate according to size
viewed as bands under UV light (fragments had fluorescent tag)
How to view a genetic fingerprint
Fragments transferred from gel to nylon membrane
Membrane incubated with fluorescently labelled DNA probe
Viewed as bands under UV light
what is genetic fingerprinting?
method used to determine an organisms genetic identity
done by electrophoresis
depends on number of VNTRs in the organisms DNA
- shown as bands under UV or radioactive light
interpretation of a genetic fingerprint
multiple fingerprints can be compared
remember fragments separate based on length (in nucleotides) and therefore VNTRs
shorter move further
if both fingerprints have a band at the same location, they have the same number of nucleotides (so same number of VNTRs)
they match
uses of genetic fingerprinting in forensic science
compare samples of DNA collected from crime scenes to samples from suspects
- DNA isolated from crime scene and suspects
- each sample replicated using PCR
- samples run on electrophoresis gel to produce genetic fingerprints
- compared to find a match
- if samples match, it can link a suspect to a crime scene
uses of genetic fingerprinting in medical diagnosis
genetic fingerprint refers to a unique pattern of alleles
can be used to diagnose genetic disorders and cancer
useful when specific mutation isn’t known or when caused by multiple mutations
because it identifies a broader genetic pattern
uses of genetic fingerprinting in animal and plant breeding
used to prevent interbreeding
- decreases gene pool
- increased risk of genetic disorders
- heath and productivity issues
used to identify how closely related two individuals are so they won’t be bred together (less related bred)
more similar fingerprints means more closely related
how is genetic fingerprinting used to determine genetic relationships?
VNTR sequences inherited from parents
- roughly half from each parent
more bands that match = more closely related
eg paternity tests - more matches = more likely to be father
how is genetic fingerprinting used to determine genetic variability? (in a population)
the more bands that don’t match = less related (more genetically different)
can find out how genetically varied a population is
the more the number of repeats varies at different places = more genetic variability
uses of genetic fingerprinting (5)
- determining genetic relationships
- determining genetic variability
- forensic science
- medical diagnosis
- plant and animal breeding
what is a primer?
short sections of single stranded DNA
base sequence is complementary to DNA fragment so it binds
allows polymerase to bind and produce the new fragments
why is mRNA used/ bacterial DNA better
mRNA and bacterial have introns removed
means bacterial DNA easier to study
fewer non-coding regions (introns)
so knowledge of genome more easily related to proteome
- less non-coding to get in the way
promotor region
tells RNA polymers where to start transcription of DNA
means gene transcribed
