Module 6.3 Flashcards
1
Q
DNA sequencing
A
a technique that allows genes to be isolated and read .
2
Q
What is early DNA research suggesting
A
by the early 1970s , the strucuture of DNA was known , as were the sequences of base triplets that coded for the various amino acids .
-However , at this time , it was difficult to work out the sequence of the nucleotide base triplets in genes .
1972 , a belgian moleuclr biologist sequen ced a gene that codes for the protein coat of a virus ms2 .
-Both scienitsits worked fromt he mRNA transcribed from the gene and not the rw DNA .
-RNA is unstable nd this hwole process was extremeleys low and only suitbale for very short genes .
1975 - FRED SANGER
3
Q
1975 - FRED SANGER
A
developed a method that ultimately allowed scientits to sequence whole genomes .
4
Q
what was Fred Sanger’s Dna sequencing approach (1)
A
Sanger’s approach was to use a single strand of DNA a s atemplate for four experiments , in separate dishes .
-Each dish contianed a solution with the four bses ATCG plus an enzyme DNA polymerase .
5
Q
what was Fred Sanger’s Dna sequencing approach (2)
A
To each dish , a modified version of one of the DNA bases was added . The bases was modified in such a way that , once incorporated into the synthesised comple,entary strand of DNA , no more bases could be added . Each modififed bases was also labelled with a radioctive isotope .
6
Q
what was Fred Sanger’s Dna sequencing approach (3)
A
As the reactions progressed , thousands of DNA fragemnts of varying lengths were generated .
-The DNA fragemnts were passed through a gel by electrophoreisis .
SMALLER GRAGMENTS TRAVELLED FURTHER SO THE FRAGMENTS BECAME SORTED BY LENGTH .
7
Q
what was Fred Sanger’s Dna sequencing approach - how was the dna fragments read
A
the nucleotide base at the end , of each fragment was read according to its radioactive label .
-If the first one base fragment has thymine at the end , then the first base would be t .
-if the two base fragment have cytoicinse is tc .
-if a THREE base fragment ends with hguanine then the seuqnece is TCG .
8
Q
how gould was Fred Sanegrs DNA sequencing approach and why
A
this method was efficent and safe .
-Sanger used it to sequen ce the genome of a phage virus ( avirus than infects bacteria ) , called phi-X174 , the first DNA based organisms to have its genome sequenced .
-He has to count off the bases one by one from the bands ina piece o gel - a very time ocnsuming and costly process . check 216 for sgtats
9
Q
How to clone DNA (1)
A
the gene to be sequenced is siolted using RESTRICTION ENZYMES from a bacterium .
10
Q
How to clone DNA (2)
A
The DNA was then inserted into a bacterial plasmid (the vector ) and then into an escherial coli bacterium host tht when culutred divided mnyt imes neabling the plasmid witht he DNA insert to be copied many times .
11
Q
How to clone DNA (3)
A
Each new bacterium contained a copy of the candidate gene . These lengths of NDA were isolated using plasmid perparation techniques and were then sequenced .
12
Q
What was the first DNA sequencing machine
A
-In 1986 , the first automated DNA sequencing mchine was developed at the Valifornia institute of technology , absed on Fred Sanger’s method .
-FLUORESCENT DYIES INSTEAD OF RADIOACTIVITY WEE USED TO LABEL THE temrinal bases .
13
Q
What was the first DNA sequencing machine (2)
A
-tHESE DYES GLOWED WHEN scanned with a laser beeam , and the light signature was idenified by computer . This method dispensed witht he needs for techhnicians to read autoradiograms .
14
Q
What is high throughput sequencing
A
In the first decade of the twenty first cenutry a vaeirty of apporaches was used to devleop fast ,c heap methods to sequence genomes ONE OF THEM IS PYROSEQUENCING
15
Q
When ws pyrosequencing developed and what is the overall process
A
This method was developed in 1996 , and uses sequencing by syntheissi , not by chain temrination as in the Sanger method .
-It invovles synthesising a single strand of DNA ,, complementaty to the strand to be sequenced one base at a time , whilst detecting by light emission , which base ws added at each step .
16
Q
stage one of pyrosequencing
A
a long length of DNA to be sequences is mechanically cu into fragments of 300-800 base pairs using a nebuliser .
17
Q
stage two of pyrosequencing
A
these lengths are then degraded into single stranded dna (ssDNA) . These are the template DNAS nd they are immbollised .
18
Q
stage three ofpyrosequencing
A
a sequencing primer is added and the DNA is then incubted with the enzymes , DNA polymerase , ATP sulfurylse , luciferase apyrase and the substrate adenosine 5’ phosphosulfate (APS) and luciferin .
-Only ONE of the four possible activated nucleotides ATP and TTP CTP and GTP is added at any one time and any light generated is detected .
19
Q
stage four of pyrosequencing a
A
one activated nucleotide (a nucleotide with two exra phosphoryl groups ), such as TTP (thymine triphosphoate , is incorporated into a complemntary strand of DNA usin the strand to be seuenced as a template .
20
Q
stage four of pyrosequencing b
A
as this happens the two extr phosphorl are released as pyrophosphate . (ppi)
21
Q
stage four of pyrosequencing s c
A
in the presence of AP , the enzyme ATP sulfurylase , converts the pyrophosphate to ATP .
22
Q
stage five of pyrosequencing d
A
in the presenc eof this ATP , the enzyme luciferase converts luciferin to oxylucifierin .
23
Q
stage six pyrosequencing e
A
this conversion gernartaes visible lgihts which cn be decretected by a camera . The amount of light genertes is proptoional to the amount of ATP avaible in th end , therfroe indicates show many of the same type o actived nucleotides were incorporated adjacently inot the complmentary DNA strands .
24
Q
stage seven of pyrosequencing
A
unincorporated activated nucleoitde are degraded by apyrase and the reactions starts again with another nucleotide .
25
what is bioinformatics
a branch of bioogy called bioinformatics has grown out of thsi research to store the huge amounts of data generated . It would have been impossible to store and analyse these data prior to computers and microchips , . Software packages are specillyd esgined for this purpose .
26
The human genome project wgat is this
scienitsts predicted human genome would conain 100k genes , and it was laucnhed in 1990 , b ut was sequenced by 203 , scienitsts were suprised the human genome contained only about 24k genes .
27
what does the whole genome sequence consits of
whole genome sequence determines the complete dna sequence of an organisms genome in the case of eukaryoutc cells tht is the genetic material of the chromosomes , m itochondria , and if plants or algae also of chlorplasts , sequenced genomes are stored in gene banks .
28
how is the human genome different to other species (1)
most of our genes have counterprts in othe rorganisms , we hve simialr genes , to other nimals , genes work well tend to be consevred ine voleution hence why we share 99 percnet of our genes with chimpanzees .
29
how is the human genome different to other species (2)
sometimes as evolution progresses some gene are co-opted to perform new tasks . Tiny changes to the gene in huamns called FOXP2 , which is found in other mammals including mice nd chimpanzees , means that in human this gene allows speech .
MAMY DIFFERENCE ACC CUZ SHARED GENES HAVE BEEN ALTERED TO WORK IN SUBTLYD IFFERNEY WAYS . sOME CHANGES OT THE REGULTORY REGIONS OF DNA DO NOT COE FOR PROTEINS HAE ALSO ALTERED THE EXPRESSION OF GENOEMS , REGULTORY ANFD CODINGG ENES INTERACT IN SUCH AYS WITHOUT INCREIDNG HTE NUMEBR OF GENES , NUMBER OF PORTEINS MADE MAY INCREASE .
30
HOW C can comparing evolutionnary relationships (1)
comparing genomes of organisms thouhgt to be closely related psecies has helped confirm their evolutionry rellationshps or has led to new nwledge about the relationship sand in some cases to certain organism being reclassififed .
31
HOW C can comparing evolutionnary relationships (2)
The DNA from bones and teeth of some extinct animals can be amplified and sequences soo that the animal;s evolutioanry hisotry can be verified .
32
why may humans not be geneticlaly similar
genes have be lsot b deltion of par of a chromsome have different lleles .
the p dna sequeqneces can differ is due to random mutaitons such as substitution .
33
where is the place in DNA , wheresubstituions can occur
they are called isngle nucleotide polymorpism , or SNPS ( pronounse snpi)
-some have no effect on the portein , some can alter a portein or lter the way a peice of RNA regultes the expression of another gene .
34
what is methylation
it palys a major role in regulating gene expression in euakryotic cells .
-Methods to map this methylation of whole humangenomes can help researchers to udnersand the development of certin diseases , for example ceertain types of cancer and whyt hey may or many nont develop in geneticlalys ismialr inidudals thi[s is clled EPIGENTICS .
35
PREDICTING THE AMINO ACID SEQUENCES OF PROTEINS
-determining the sequence of amino cids within a portein is laboriuos and time ocnsuming . HOwever , if researchers have the ogranims;s genome sequencedd and know which gene codes for s aspeicif cportein , b usign kwnodlege of w hicch base triplets code for which aminoa icds , they can detemrine the priamrys trucutre of proteins . The reserchers need to now which part of the gene codes fo rthe exons and which does for intorns .
36
what is synthetic biology
syntehtcic an interdisiplianry science concerned with designing and builing useful biolog devices and syems .
-It encompasses biotehcnology evolutionary biology , molecular biologgy , syems biology and biophsycis . Its ultimae goals may e build engineered biolgoical sysems that store and process informaion , provide food mintain human health and ehnace the enviornemnt .
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aspects of syntheitc biology
biofules
-biomedicine
-chemistry
-computer science
-engineering science
-physics .
38
application of synthetic biology - information storage
scientists can encode vast amounts of digital information onto a single strand of synthetic DNA . One project has encoded the complete works fo William Shakespee onto a strnad of sytnewhtic dna
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applications of syntehtic biology - productions of medicines
escherial coli nd yeas egenticial engineered to produce antimalrial durg .
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applications of annaotechnolgoy - nanotechnology
materials can be produced for nanotechnology e.g amyloid fibred to making biofils ofr function uscha s adheison .
40
isssues of syntehitc biology
synthetic biology raises issues of ethics and biosecurity .
--Extensive regultions are already in place due to 30-40 years of using geneitcally modififed organisms .
41
issues of syntheitc biology (2)
-There are many advisory panels and manys cienfitic papers have been writeen on how to manage the riss .
-Syntheitc biology is not aout makign syntheitc life forms froms crach , but is about a potenit for new systems with reward and associated riss to be amanged .
42
development of dna profiling ALEC JEFFREYS - locating tandem repea sequences wha arre these
they are repetitive segements of DNA that do not code for proteins .
sometimes re highly variable and called
variable number tandem repeats. (VNTRs)
-they all eature same core sequence sequence has an x where it can be onl one of four nucleotiedes .
43
stage one of dna profiling
DNA is oobtained from the individual - either by mouth swab , from saliva , on a oothbrush , from blood or hair or in the case of ancient remians from bone .
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stage two of dna profiling
the DNA is then digested with restriction enzymes . These enzymes cut the DNA at specific recognition sites . They will cut it into fragments , which will vary in sie from individual to individual .
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stage three of dna profiling
the fragments are separated by gel eelectroophorwesis and stained . Larger fragments travel the shortest distance in the gel .
46
stage four of dna profiling
a banding pattern can be seen .
47
stage five of dna profiling
the DNA to which the indiviudal;s is being compared is treated with the same restriction enzymes and also subjected to electrophoresis .
48
stage six of dna profiling
the banding patterns of the DNA samples can then be compared .
49
what is the first method invovleing restriction fragment length - it is no longer used
polymorphism - method is laborious so therefore no longer used
50
what is used instead of polymorphism
short tandem repeats , seuqeunced of DNA are used , these are highly variable , short repeating length of DNA , hte exact number of STRS varies from person to person .
STR sequencies ae separated by electrophoreisis each STR is polymorphic , but the number of alleles in the gene pool for each one is small , str is present in beween 5 and 20 percent of idnivudals so the chance of two people sharing str , sequneces at all loci si blah blah blah .
51
sensitivity of str analysis
the technique is ver sensitive and even a trace of DNA left when someone touches an object cn produce a result samples must be treated carefully to avoid contaminaition .
-DNA can be stored for man yers if a cirme case is unsovled it can then be later be used to assess new evidence .
52
one application of dna profiling - FORENSIC SCENCE
DNA profiling has transformed forensic science . Not only has it rought about convicitionns , and established the innocnence of amny suspects and wrongly convicted .
remains body parts of people from ttacks ,
profiles of descedants for world wars .
53
another application of dna profiling - amtenirty paternity tests
genetic info half from mother and half from father , comparing the dna profile can idneitfy amtenirty or patenrity .
54
another applicaiton of dns profiling - analysis of disease
protein electrophoriesis can detect the type of haemoglobin present and aid diagnosis of sickle cell anaemia . A varying number of repeat sequences dfor a conditions such as Huntington disease can be detected bye electrophoresis .
55
learning tip about dna profiling
dna proifiling does not loo at egens that code for proteins ,w e are far too similar to each other in this respect
56
what is pcr (polmerase chain reaction )
a biomedical techology in molecuar biology that can amplify a short length o fDNA to thousands of millions of copies .
57
What is PCR (polymerase chain reaction ) PCR
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies .
58
What is PCR (polymerase chain reaction ) PCR
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies .
59
Modern dna profiling is good as it can obtain results from as few as five cells , buoy what is the problem
If it is a surface frequently touched by oriole k it could transfer innocent oeilkes DNA into the hands of a criminal , ,
60
What are the facts the PCR ( the artificial replication of DNA) relies on (1)
- DNA is made of two anti parallel backbone strands .
_ Each strand of DNA has a 5’ end and a 3’ end .
- DNA grows only from the 3’ end .
61
What are the facts the PCR ( the artificial replication of DNA) relies on (2)
- base pairs pair up according to complementary base pairing rules A with T and G with C .
62
First way PCR differs from DNA replication
- Only short sequences up to 10k base pairs of DNA can be replicated not entire chromosomes .
63
Second way PCR differs from DNA replication
It requires the addition of primer molecules to make the process start .
64
Third way PCR differs from DNA from DNA replication
A cycle of heating and cooling is needed to separate the DNA strands , bind primers to the strand and for thr DNA strands to be replicated .
65
Why was the PCr process lengthy before
At first the process was quite time consuming as the DNA was heated to denature it , and then cooled to around 35 degrees k to anneal the primers and allow the DNA polymerase to work . Later DNA , polymerase was obtained from the thermophikic bacterium . Thermiohikjs aquatics . This enzyme is caked TAQ polymerase and is stable at high temperatures .
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Step one of PCR
The sample of DNA is mixed with DNA nucleotides , primers magnesium ions enzyme TAQ DNA polymerase .
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Step two of PCR
The mixture is heated to around 94-96 degrees to break the hydrogen binds between complementary nucleotide base pairs and thus denature of thr double stranded DNA into two single strands of DNA .
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Step three of PCR
The mixture is cooled to around 68 degrees , so that the primers can sneak (bind by hydrogen binding ) to one’s end of each single strands of DNA . This gives a small section of double stranded DNA at the end of each sing,e stranded molecule ,
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Step four of PCR
The TAQ DNA polymerase enzyme Melodie’s can now bind to the end where the is double stranded DNA into. Tara polymerase is obtained from a bacterium that lives at high temperatures ; 72 degrees is the optimum temperature for this enzyme .
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Step four of PCR
The TAQ DNA polymerase enzyme Melodie’s can now bind to the end where the is double stranded DNA into. Tara polymerase is obtained from a bacterium that lives at high temperatures ; 72 degrees is the optimum temperature for this enzyme .
71
Step 5 of PCR
The temperature is raised to 72 degrees which keeps the DNA as single strands .
72
Step 6 of PCR
The TAQ DNA polymerase catalyses the addition of DNA nucleotides , to the single stranded DNA molecules starting at the end with the router and proceeding in the 5’ to 3’ direction .
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Stage 7 of PCR
When the TAQ DNA polymerase reaches the other end of the DNA molecule , then a new double stranded kf DNA has been generated .
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Stage 8 of PCR
The whole process begins again and is repeated for many cycles .
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Give one application of PCR -tissue typing
Donor and recipients tissues can be ruled prior to transplantation to reduce the risk of rejection of the transplant .
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Application of PCR - detection of oncogenes .
If the type of mutation involved in a specific patient’s cancer is found , then the medication may be better tailored to that patient .
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Applications of PCR_ detecting mutations
A sample of DNA is analysed for the presence of a mutation thst leads to a genetic disease . Parents can be tested to see if they have a recessive allele for a particular gene , feta, cells may be obtained , from the mothers blood stream for prenatal genetic screening duirng IVF treatment one cell from an eight cell embryo can be used to analyse the fatal DNA before implantation .
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Application of PCR-identifying viral infections
Sensitive PCR tests can detect small quantities of viral genome amongs the host cells DNA . This can be used to verify for example HIV hepatitis C infections .
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Applications of PCR-Monitoring the spread of infectious disease
The spread of pathogens through a population of wild domestic animals or from animals to human populations can be monitored and the emergence of new more virulent sub types can be detected .
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Application of PCR - forensic science
Small quantities of DNA can be amplified for DNA profiling to identify criminals or to ascertain over engage .
81
Application of PCR -Research
Amplifying DNA from extinct ancient sources such as Neanderthal or wikky mammoth bones , for analysis and sequencing for extant organisms , tissues or cekks can be analysed time fund it whucgbgeens are switched in or off .
82
What is electrophoresis
Process used to separate proteins or DNA fragments kf different sizes ,
83
What technique does gel electrophoresis
Use
The technique uses an agarose gel plate covered by a buffer solution . Electrodes are placed in each end kf the tank so that , when it is connected to a power supply an e,extricate current can lass through thr
84
What technique does gel electrophoresis use (2)
gel . QDNA , has an overall negative charge due to its many phosphate groups , ain’t the fragments migrate towards the anode (positive electrode). Fragments of DNA all have similar surface charge regardless of their size ,
85
Stage one of gel electrophoresis
1. The DNA samples are first digested with restriction enzymes to cut them , at specific recognition sites , into fragments . This is carried out at 35-40 degrees and may take up to an hour .
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Stage 2 of gel electrophoresis
While the restriction enzymes are cutting the DNA , the tank is set up . The agarose gel is made up and perused into the central region of the tank , whilst combs are in place at Ken end . Once the gel is set , buffer solution is added so that the gel is covered snd the end sections of the tank contain buffer solution . Now the comb can be carefully removed leaving wells at one end kf thr gel .
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Stage three of gel electrophoresis
A loading dye is added to the tubes containing the digested DNA.
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Stage four of gel electrophoresis
The digested DNA plus loading dye is added to wells in the electrophoresis gel plate covered. To do this , a pipettes is used and this is held , in the buffer solution m just above one of the we,,s . The loading dye is dense and carriers
The DNA down into the well . The pipettes should not be placed right into the well , otherwise you might pierce the bottom kf the well .
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Stage 5 of gel electrophoresis
Once all the wells have been loaded with the different DNA samples , the electrodes are put into place and connected to an 18V battery . This is then left to run for up to 6-8 hours .
90
Stage 5 of gel ectrophroesis 5 voltage
lternatively , a higher voltage power pack can be shed and gel run a much shorter time less than 2 hours , do do not use a higher voltage unless the current is limited to 5mA or less , otherwise there is a risk kf severe electric shock from the eekcrides or gel .
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Stage 6 of gel electrophoresis
The DNA fragments move through the gel at different sooedsm. Smaller fragments travel faster k so in a fixed period they travel further .
92
Gel electrophoresis stage 7
At the end kf the period , the buffer solution is poured away and a dye is added to the gel . The dye adheres to the DNA and stains the fragments .
93
Separating proteins - principle
The principle for separating proteins is the same as for separating DNA fragments, but is often carried out in the presence of charged detergent such as sodium sidedly surface equallisss the surface charge on the molecules .
snd allows the proteins to seps4ste as Rhett mice thriugh the gel , according to their molecular mass . In some cases the protein can be separated according to the mass and then without SDS according to their surface charge .
94
Separating proteins
Technique can be used to type of haemogolobin proteins for diagnosis of cois nditions such as ;
is
-Siickle cell anaemia , where the patient has ahemoglobin S and not the normal haemoglobin A .
-aaPLASTIC ANAEMIA thalassaemia , and leukamia where the patients hv higher than norml amounts of fetal heamoglobin , haemoglobin f nd lower thn normal amounts of haemoglobin a .
95
what is a dna probe
a dna probe is a short 50-800 nucleoitde single stranded length of DNA tht is complementaaty to a section of the DNA being ivestigated .
96
97
What could a dna probe be labelled with
A radioactive marker using p32 , in one of the phosphate groups in the probe strand . Once the probe has annealed (bound) by complementary base pairing to thr price of DNa it can be revealed by exposure to photographic film .
98
Second way of dna. Probe being labelled
A fluorescent marker that emits a colour on exposure to the UV light . Flourosvent markers may also be automated DNA sequencing .
99
One way of locating DNA probes
To locate a specific gene needed for use in genetic engineering .
100
Second way of locating DNA probes
To identify the same gene in a variety of different genomes from different species when. Conducting genome comparison studies .
101
Third away of using dna probes
To identify the presence or abcsne of a specific allele for a particular genetic disease or that gives Buktt to a particular condition .
102
What are microarrays
Scientists can place a number of different probes Ona. Fixed surface known as dna microarray applying the rna under investigation to the surface can reveal the presence of mutate d alleles that match the fixed probes because the sample Adana will anneal to any complementary fixed probes .
103
What do you need to do to the sample of DNA for it to be Syed i. Microarrays .
The sample DNA must first be broken into smaller fragments and it may also be amplified using the polymerase chain reaction PCR . A DNA microarray can be made with fixed probes specific for certain sequences found in mutated alleles that cause genetic diseases in the well .
104
Hownnaybabdna microarray be used
Reference and test DNA samples are labelled with fluorescent markers . Where a test subject and reference markers both bind to a particular probe k the scan reveals fluorescence of both colours indicating the prescebce of the particular sequence in the test DNA.
105
What type of gel does gel electrophoresis use
Uses agarose gel it is similar to agar on which we grow bacteria but not exactly the same .
106
What is dna liagse
Enzyme that catalyses the joining of sugar and phosphate groups within DNA.
107
What is electroportarion
Method for introducing a vector with novel gene into a cell a pulse of electricity makes the recipient cell membranes more ProRes .
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What are plasmids
Small loops or DNA in prokaryotic cekks .
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What are is recombinant DNA
A composite dna molecule created in vitro joining foreign DNA with vector molecule such as a plasmid .
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What are restriction enzymes
Endonuc,ease enzymes that cleave DNA molecules at specific recognition sites .
111
What is a vector
In green technology , anything that can insert DNA into a host organism examples of such vectors include plasmids viruses and certain bacteria .
112
Why is genetic engineering also known as recombinant DNA technology
Because it involved combining DNA from from different organisms . It is also called genetic modification . ors .
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Stage one of genetic engineering
The required gene is obtained .
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Stage two of genetic engineering
The copy of a gene is placed inside a vector .
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Stage three of genetic engineering
The vector carries the gene into a recipient cell l
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Stage four of genetic engineering
The recipient expresses the novel gene .
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Technique in genetic engineering - obtaining a gene
mRNA can be obtained from cells where the gene is being expressed . An enzyme reverse transcriptase can then catalyses the formation of a single strand of complementary DNA cDNA and using thr mRNA as a template . The addition of primers and DNA k polymerase can cDNA into a double stranded length of DNA , whose base sequence codes for thr original protein .
118
Obtaining the required gene (2)
If scientists know the nucleotide sequence of yhe gene the. The gene can be synthesised shunt an automated polynucleotide sequence .
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Obtaining thr rehired gene (3)
If scientists know the sequence of the gene , they can design polymerase chain reaction primers to ampmloft the gene from the gene:if DNA .
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Obtaining the gene (4)
A DNA probe can be hsed to locate a gene within the genome snd yhe gene can then be cut out using restricutin enzymes .
121
Second technique - placing the gene info a vector ()
Plasmids can be bobtailed from organisms such as bacteria snd mixed with restriction enzymes that will cut the plasmids at specific recognition sites .
122
Second technique - placing the gene info a vector (2)
Thr cut plasmid is exposed unpaired nucleotide bases , callled sticky ends .
123
Second technique - placing the gene info a vector (3)
If free nucleotides vases complementary to the sticky ends of the plasmid are added to the ends of the gene can be inserted then the gene snd cut plasmid should anneal (bind) . DNA Logan’s enzymes catalyses the annealing .
124
Second technique - placing the gene info a vector (4)
A gene may be sealed into an attenuated (weakened ) virus that could carry it into a host cell .
125
Why is getting the vector into a recipient cell difficult
As DNA does not easily cross the recipe takes xell plasma membrane , various methods can be used to aid the process .
126
First way of getting vector into the recipient cell - heat shock treatment
If bacteria are subjected to alternating periods of cold snd heat , in the presence of calcium chloride m their walls and membranes , will become more porous and allow in the recombinant vector . This is because the positive calcium ions surround the negatively charged parts of the DNA. Molecules and. Phospholipid in the cell membrane thus reducing repulsion between thr foreign DNA and the host cell membranes .
127
Second way of getting vector into recipient cell electroportation
A high voltage pulse is applied to the cell to disturb the membrane .
128
Third way of getting vector into the reidpnet cell - electro fusion
Electrics, fields help to introduce DNA into cells ,
129
Fourth way of getting vector into recipient cell transfection
DNA. An be packaged into a bacteriophage which can then transfect thr host cell .
130
Fourth way of getting vector into recipient cell transfection
DNA. An be packaged into a bacteriophage which can then transfect thr host cell .
131
Fifth way of getting vector into recliner cell T1
Recombinant plasmids are inserted into the bacteriaagora bacterium tunefaciens which infect some plants and naturally inserts its genome into the host cells genome .
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4 direct method of introducing gene into recipient
If plants are not susceptible to Atumefaciens , then direct methods can be used to. Small pieces of gold or tungsten are coated with the DNA and shot into the plant cells . This is called a gene gun .
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Reverse transcriptase
Retroviruses , such as HIV which contain RNA that they inject into the host genome , have reverse transcriptase enzymes that catalyses the production of cDNA ( complementary DNA) , using their RNA as a template . This is the reverse of transcription . These enzymes are useful for genetic engrineering as outlined above .
134
Restriction enzymes
Bacteria and archaea have restriction enzymes , called restriction endonuclease , to protect then from attack by phage viruses . These enzymes cut up the foregin viral DNA , by a process called restriction , preventing the viruses from making copies of themselves . The prokaryotic DNA. Is protected rpm the action of these endonuclewses by being methylated at the recognition sites .
135
How are the restrictio endonucleases named
Restriction dmdonucleases are named according to thr bacterium from whuch they have been obtained . The first one used was ECOT1 it was obtained from E.colili and was restriction endonculease number 1 .
136
How are restrictions enshmes is useful to molecular biology and biotechnology
Useful as molecular scissors as they recognise specific sequences within a length of DNA and cleave fhe molecule there . Some make a staggered cut leaving sticky others make a cut that orieyce blunt ends .
137
What are Logan’s enzymes
DNA ligand enzymes are used n molecular biology to join DNA fragments . It data,she’s condensation reaction that join the sugar groups and phosphate grihos of thr DNA backbone . These enzymes catalyse such reactions during DBA eel,Icarian in cekks and are a,so hsed tin the PCR
138
Insulin from GM bacteria (1)
Scientists can obtain mRNA from beta cells of islets of Langeehans in the human pancreas where insulin is made .
139
Insulin from GM bacteria (2)
Adding se transcriptase enzyme molecules makes a single Serena’s of cDNA and treatment with DNA polymerase makes a double strand. The gene .
140
Insulin from GM bacteria (2)
Adding reverse transcriptase enzymes makes a single strand of cFNA and treatment with DNA polymerase makes a double strand -the gene .
141
Insulin from GM bacteria (3)
Mow with the be,l kf ligament enzymes , thr insulin bene can be inserted into plasmids , extracted from E.coli bacteria . These are now callled recombinant plasmids , as they contain insert DNA .
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Insulin from GMbactedia (4)
E.coili bacteria are mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions so that they take up the plasmids ,
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How are genetically modified bacteria are cultured in large numbers to do what
Check figure 2 page 230
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How are transgenic bacteria ensured they are Sade
They have a gene knocked out whuch means they cannot synthesis is a particular nutrient . They survive in the laboratory where they are given that nutrient in their forethought medium but willl not survive outside of the laboratory .
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Benefit of modifying organisms E.coili
GM microorganisms can make human insulin to treat all diabetic (this was not possible using pig insulin ), and human growth hormone to treat children with pituitary dwarfisms .
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Potential hazards of e.coili
Microrganjsms con,d be hsed escape into the wild and transfer marker genes for antibiotic resistance to other bacteria . However the GM bacteria are also modified sonrheycannot synthesise an essential nutrient and therefore cannot live outside the lab .
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Potential benefits of plants by tobacco and maids
In 1985 tobacco plants were genetically modified to produce the toxins normally produced the toxin normally produced by a bacterium , ingenious . This toxin has been used by organic farmers as a pesticide , as it is toxic to insects . Because the backs, gene but was inserted into Simeon crib olanrs , the gym olanrs produced the toxin eliminating the need to spray kf around the environment possibly contaminating other organisms .
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Potential hazards kbscco and be mwjze
Bt is toxic to monarch bugged,Joe’s , hwiever , these butterflies do not take nectar from tobacco olanrs or maize plants in the wild , the feed on milkweed desojre many thousands kf hectares of ,and in the USA being oak fed by cries the monarch butterfly has continued kf thrive .
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Soya beans potential benefits
Gm soya beans eejssganr to herbicide weee produced so Rhayader weeds competing with the soya plants could be killled with herbicides ,
150
Soya beans potential hazards
Possible risks include the potential, for the bene for herbicide resistant to pass into sjeoeweeds this does not aloeae to nabe hallwens rk date .
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What is germ line therapy
Gene therapy by inserting functional alleles into gamete’s or zygotes ,
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Somatic cell gene therapy
Gene therapy by inserting duncfuinak Akko’s unfit bidy cekks ,
153
What is fhe principle of gene therapy
The basis principle of raspy is to insert a functional alleles into gamete’s of a particular gene into cells that contain only mutates and non only mutates and non of a particular gene gene into cells that contain only mutated and non functioning alleles do Rhayader gene . If the inserted allele is expressed then the individual willl produce a functioning protein snd j longer have symptoms associated with the genetic disorder ,
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What are liposomes
Lengths of DNa san be paskagws within small Albee’s ld lipid bilateral to make lipososokss . If these are placed into an aerosol inhaler snd sprayed into the noses of pareinrd some will pass through the plasma memenae kf cekks lining the respiratory react ,
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Problems with viruses
Viruses even though not virulent may Siri provoke an immune response or iinfmalmmaorry response in patients .
The patients may become immune to the virus making subsequent deliveries difficult or impossible .
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Problems with viruses (2)
The viruses may insert the allele into the patients genome in a location that disrupts in a gene involved in regulating cell division increasing the risk of cancer .
- Themfiru may insert fhe alleles into the patients genome in a location that dispute the regulation of fhe expression of other genes .
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Artificial chromosomes
Research is being carried out into the possibility of inserting genes until an artificial chromosomes that Wouk’s do exist with others 46 chromosomes in the target cekks ,
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What is gene line rheeaoyn
Germ line therapy involves atendaifng the genome of gametes or zygotes .
Not into will ryendekks of idnicdual be altered m their offspring may also inherit thr foreign alleles . Thus this type of therapy. Has the potential to change fhe generic makeup of many descnednsrs of the original patient none of whom would have given consented . Also concerns about how the egene may be inserted x they Amy find rheum way Iranian kicarjin thst ckukd disrupt the edoressjin kf refuksfjin kf other genes or jcndease fhe risk kf cancer .
