8b (genome/gene tech) Flashcards
why do you need to chop DNA into smaller pieces before sequencing?
only works on fragments of DNA
smaller pieces are sequenced then put back in order to give sequence of whole genome
why is it relatively easy to determine proteome of simple organisms?
why is this useful?
they dont have much non-coding DNA
can be used in medical research and development eg identifying protein antigens on surface of disease-causing bacteria/ viruses to help develop vaccines
can also monitor pathogens in disease outbreaks to help manage and identify antibiotic resistance factors
why is it more difficult to translate genome into proteome in complex organisms?
contain large sections of non-coding DNA
contain complex regulatory genes which determine when genes are on/off
hard to find the bits that code for proteins among the other parts
old sequencing methods vs new
old- labour intensive, expensive, small scale
new- automated, cost-effective, large sale, quicker
what is the aim of the genome project?
improve understanding of genetic factors in human disease so new ways to diagnose and treat illness can be developed
what is recombinant DNA technology?
what are the steps?
transferring fragment of DNA from one organism to another. genetic code is universal and transcription/ translation mechanisms are similar so transferred DNA can produce protein in cells of recipient
1. making DNA fragments
2. amplifying DNA
3. checking if the cell has been transformed
what are transgenic organsisms?
organisms that contain transferred DNA
what are the 3 methods of producing DNA fragments?
reverse transcriptase
restriction endonuclease
gene machine
using reverse transcriptase to produce DNA fragments
most cells only contain 2 copies of each gene so difficult to obtain DNA fragments containing target gene
cells that produce protein coded for by target gene will contain many mRNA molecules complimentary to the gene so mRNA easier to obtain
mRNA used as template to make lots of DNA using reverse transcriptase
DNA produced is called complimentary DNA (cDNA)
- mRNA isolated from cells
- mixed with free DNA nucleotides and reverse transcriptase
- rt uses mRNA as template to synthesise new strands of cDNA
also, plasmids cant splice introns
using restriction endonucleases to produce DNA fragments
some sections of DNA have palindromic sequences of nucleotides- consist of antiparallel base pairs
restriction endonucleases recognise specific palindromic sequences (recognition sequences) and cut the DNA at there places
diff re cut at diff specific recognition sequences as shape of recognition sequence is complimentary to enzymes active site
if recognition sequences are present either side of DNA fragment, restriction endonucleases can be used to separate it from rest of DNA
DNA sample incubated w specific restriction endonuclease, cutes fragment by hydrolysis. sometimes leaves sticky ends- used to bind DNA fragments together
what are sticky ends?
small tails of unpaired bases at each end of the fragment
using gene machine to produce DNA fragments
- can synthesise fragments from scratch- no templated needed so sequence doesnt have to exist naturally
- sequence is designed
- first nucleotide in sequence is fixed to some sort of support eg bead
- nucleotides added step by step in correct order, in cycle of processes that includes adding projecting groups (make sure nucleotides are joined at right points to prevent unwanted branching)
- short sections of DNA (oligonucleotides) are produced. theyre broken off from support and projecting groups removed. oligonucleotides are joined to make longer fragments
what does amplifying DNA fragments mean?
What are the 2 methods?
making more copies of DNA fragment through gene cloning
- in vivo- gene copies made within living organism
- in vitro- gene copies made outside of living organism using PCR
In vivo cloning part 1- making recombinant DNA
- vector (used to transfer DNA into cell eg plasmids/ bacteriophages) DNA is isolated
- vector DNA cut open using same restriction endonuclease used to isolate DNA fragment containing target gene
- vector DNA and DNA fragment are mixed together with DNA ligase which joins the sticky ends together in a process called ligation
- the new combo of bases in DNA (vector DNA and DNA fragment) is called recombinant DNA
why do you use same restriction endonuclease to cut open vector DNA as you use to isolate DNA fragment?
so sticky ends of the vector DNA are complimentary to stick ends of DNA fragment containing the gene
In vivo cloning part 2- transforming cells
the vector with the recombinant DNA is used to transfer the gene into host cells
host cells that take up vectors containing the gene of interest are said to be transformed
if plasmid vector used, host cells have to be persuaded to take in the plasmid vector and its DNA (need to make their membrane more soluble chich is done by adding ice cold calcium chloride than heat shocking)
with bacteriophage vector, bacteriophage infects host bacterium by injecting its DNA into it. the phage DNA with target gene then integrates into bacterial DNA
In vivo cloning part 3- identifying transformed cells
only around 5% of host cells take up vector and its DNA so need to be able to identify which have been transformed. Marker genes can be used
- marker gene inserted int vectors at same time as gene to be cloned so any transformed hosts will contain the gene to be cloned and the marker gene
- host cells grown on agar plates and each cell divides/ replicates its DNA creating colony of cloned cells
transformed cells produce colonies where all cells contain the cloned gene and marker gene
marker gene can code for antibiotic resistance- host cells grown on agar plate containing specific antibiotic so only transformed cells that have the marker gene survive. or, marker gene can code for fluorescence so agar plate under UV- only transformed cells will fluoresce - identified transformed cells are allowed to grow more producing lots of copies of cloned gene
how is replica plating used as a marker to see if a gene has been taken up?
1) bacterial cells cultured by spreading them on a nutrient agar plate
2) each separate cell on plate will grow into genetically identical colony
3) small sample of each colony transferred to secondary plate in same position as colonies on original plate
4) replica plate contains diff antibiotic (tetracycline) against which the antibiotic-resistance gene will have been disabled if the new gene has been taken up
5) colonies killed by tetracycline must be the ones that have taken up the required gene
6) colonies in same position on og plate are the ones that posses required gene. these colonies are made of bacteria that have been genetically modified and have been transformed
how are fluorescent markers used as a marker to see if a gene has been taken up?
1) gene transplanted into centre of green fluorescent protein gene
2) any bacterial cell that has taken up the plasmid wo the gene that is cloned will not be able to produce GFP
3) the cells that have taken up gene will not fluoresce
4) as bacterial cells w desired genes are not killed, theres no need to replica plating
5) results obtained by viewing cells under microscope and keeping those that dont fluoresce
describe polymerase chain reaction/ PCR (in vitro cloning)
- reaction mixture set up that contains DNA sample, free nucleotides, primers and DNA polymerase
- DNA mixture is heated to 95c to break the hydrogen bonds between the 2 strands of DNA.
Its then cooled to 55c so the primers can bind (anneal) to the strands - the reaction mixture is heated to 72c so DNA polymerase can work. it lines up free DNA nucleotides alongside each template stand and joins the nucleotides together. specific base pairing means new complimentary strands are formed
- 2 new copies of the fragment of DNA are formed and one cycle of PCR is complete.
cycle starts again- heated to 95c and this time all 4 strands used as templates
what are primers?
what do they do?
short pieces of DNA that are complimentary to the bases at the start of the fragment you want
- add promotor regions and terminator regions so DNA polymerase knows where to start and stop (so correct protein produced)
- prevent strands from joining back together
what is genetic engineering?
transforming microorganisms, plants and animals using recombinant DNA technology
transformed microorganisms can be made using same tech as in vivo cloning
how can transformed plants be produced?
gene that codes for desirable protein is inserted into plasmid
plasmid added to bacterium which is used as a vector to get gene into plant cell
if right promotor region added, transformed cells will be able to produce desired protein
how can transformed animals be produced?
gene that codes for desired protein inserted into early animal embryo/ egg cell
embryo- body cells of resulting transformed animal contain gene
egg- offspring of the female will contain the gene
why is taq polymerase used?
it comes from bacteria that live in hot springs- can withstand high temps without denaturing
benefits of transformed organisms in agriculture
transform crops to give higher yields/ more nutritious so can reduce risk of famine and malnutrition eg golden rice- more vit A
can make them pest/ drought resistance
need fewer pesticides so reduce cost/ environmental problems from using chemicals
benefits of transformed organisms in industry
can produce enzymes in large quantities for less money
benefits of transformed organisms in medicine
making drugs and vaccines eg insulin
can be made quickly and cheaply in large quantities- reduces cost so available for more people
concerns about transformed organisms in agriculture
monoculture (planting one type of transformed crop), makes whole crop vulnerable to same disease as theyre identical. also reduces biodiversity
superweeds- resistant to herbicides- happen if transformed crops interbreed with wild plants- cause uncontrolled spread of recombinant DNA
organic farmers’ crops may be contaminated by wind-blown seeds from nearby modified crops- cant sell their crops as organic
concerns about transformed organisms in industry
may not have a proper choice about consuming gm crops- lack of proper labelling
process of purifying proteins (from gm organisms) could introduce toxins into food industry
large biotech companies grow and get more powerful, smaller companies forced out of business
concerns about transformed organisms in medicine
companies that own genetic engineering tech may limit use to tech that could save lives
could be used unethically- designer babies
ownership issues (once genetic material removed, does donor or researcher own it?)
how does gene therapy work?
involves altering mutated alleles inside cells to treat genetic disorders/ cancer
caused by 2 mutated recessive- add working dominant allele
mutated dominant- silence it by adding DNA to middle so no longer works
involves inserting DNA fragments into person’s og DNA using vector
what are the 2 types of gene therapy?
somatic- altering alleles in body cells. doesnt affect sex cells so offspring can still inherit disease
germ line- altering sex cells. every cell of any offspring produced from these cells will be affected by gene therapy and wont suffer from disease. illegal in humans
what are the ethical issues surrounding gene therapy?
tech could be used in other ways than medical treatment eg treating cosmetic effects of ageing
potential to do more harm than good- risk of overexpression of genes
only around 5% of host cells take up vector and its DNA. what are the 3 things that can happen.
- host takes up vector and DNA
- host closes up without taking up DNA
- DNA creates its own ring
what are DNA probes used for?
locate specific alleles of genes or see if a person’s DNA contains mutated allele that causes genetic disorder
what are DNA probes?
short, single strands of DNA
specific base sequence complimentary to base sequence of part of target allele
means that it will hybridise to target allele if present in DNA sample
label attached so can be detected (radioactive/fluorescent)
how are fluorescently labelled probes used?
sample of DNA digested into fragments using restriction enzymes then separated using electrophoresis
separated DNA fragments are transferred to nylon membrane and incubated w fluorescently labelled DNA probe
if allele present, DNA probe will hybridise to it
membrane exposed to UV light and if gene present, there will be a fluorescent band
how do you screen for multiple genes?
probe can be used as part of DNA microarray (glass slide with microscopic spots of diff DNA probes attached in rows)
- sample of fluorescently labelled human DNA is washed over the array
- if labelled human DNA contains any DNA sequences that match any of the probes, it will stick to array
- array washed to remove DNA that hasnt stuck then visualised under UV light
- labelled DNA will fluoresce. any spots that fluoresce means the person’s DNA contains that specific allele
what are the uses of screening with DNA probes?
- help identify inherited conditions
- determine how a patient will respond to a specific drug
- help identify health risks
- however, some think it could lead to discrimination from insurance companies/ employers if someone more at risk of developing condition
what is genetic counselling?
advising patients and relatives about risks of genetic disorders
advise people about screening and explaining the results
screening can see if someones a carrier of mutated allele, the type of mutated allele and most effective treatment
if results are positive, counselling used to advise patient on options/ prevention treatments eg angelina jolie mastectomy
what is personalised medicine?
results of genetic screening can be used to personalise medicine
diff people respond to same drug in diff ways so some more effective than others
can find the most effective for you
what are variable number tandem repeats (VNTRs)?
base sequences that dont code for proteins and repeat next to each other over and over
number of times the sequences are repeated differs person to person so length of the sequences in nucleotides differs too
what is genetic fingerprinting?
repeated sequences occur in lots of places in genome
no of times a sequence is repeated (so no of nucleotides) at diff places in the genome can be compared between individuals
why is the chance of 2 individuals having the same genetic fingerprint very low?
the chance of 2 individuals having same number of VNTRs at each place theyre found in DNA is very low
what are the steps of genetic fingerprinting?
1) PCR used to make DNA fragments
2) separation of DNA fragments by gel electrophoresis
3) analysis of the genetic fingerprints
step 1 of producing genetic fingerprints
(PCR used to make DNA fragments)
DNA obtained eg from blood
PCR used to make many copies of the areas of DNA that contain VNTRs
primers used to bind either side of these repeats so whole repeat is amplified. diff primers used for each position under investigation
end up with DNA fragments where length in nucleotides corresponds to no of repeats person has at specific position
fluorescent tag added to all DNA fragments so can be viewed under UV light
step 2 of producing genetic fingerprint (separation of DNA fragments by gel electrophoresis)
to separate out DNA fragments, DNA mixture is placed in a slab of gel and covered in buffer solution that conducts electricity
electrical current is passed through gel- DNA fragments are - so move towards + at far end of gel
shorter DNA fragments move faster and travel further through the gel, so DNA fragments separate according to length
this produces a pattern of bands
step 3 of producing a genetic fingerprint
(analysis of genetic fingerprints)
after gel has been running long enough, equipment turned off and gel is placed under UV light - DNA fragments can be seen as bands
these bands make up genetic fingerprint
DNA ladder may have been added to one well- this is a mix of DNA fragments of known length that allows you to work out length of other bands on the gel
2 genetic fingerprints can be compared eg if both fingerprints have band at same location on the gel it means they have the same number of nucleotides and so same no of VNTRs at the place- they match
what are the uses of genetic fingerprinting?
determining genetic relationships- we inherit VNTR base sequences so more bands that match, more closely related. looking at just female line of descent, look at mitochondrial DNA. male- just look at Y chromosome
determining genetic variability- more bands that dont match, more genetically diff so can compare no of repeats in several places to see how varied a pop is
forensics- compare samples of DNA from hair, blood, semen etc with DNA from suspects. collect sample, PCR, gel electrophoresis, genetic fingerprints
medical diagnosis- useful when specific mutation isnt known/ several mutations could have caused it (identifies broader, altered genetic pattern). eg some tumours, Huntington’s, CF
animal and plant breeding- prevents inbreeding and can prove pedigree (who an animals parents/ descendants are)- animals with good pedigree sell for more
describe separation by gel electrophoresis
1) DNA fragments placed on agar gel and voltage applied across
2) resistance of gel means larger fragments move slower
3) over fixed period, smaller fragments move further than larger ones
4) DNA fragments of diff lengths are separated
5) if DNA fragments labelled (radioactive probe), their final positions can be determined by placing sheet of X-ray film over agar gel for several hours
6) radioactivity from each of DNA fragments exposes the film
7) only DNA fragments around 500 bases long can be sequenced in this way
8) larger genes and whole genomes would have to be cut into smaller fragments by restriction endonucleases
+ and - of in vitro cloning
+
very fast
doesnt require living cells
_
cant be used to transform other organisms
requires a very pure sample
at one time, about 20% cloned inaccurately
copies whole DNA sample not just specific gene
+ and - of in vivo cloning
+
useful when introducing a gene into another organism
no risk of contamination
very accurate
cuts out specific genes
produces transformed bacteria that can be used to produce large quantities of gene products
_
takes many days/weeks to produce same quantity of DNA
complex culturing techniques
