Module 6.3 Flashcards
DNA sequencing
a technique that allows genes to be isolated and read .
What is early DNA research suggesting
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
1975 - FRED SANGER
developed a method that ultimately allowed scientits to sequence whole genomes .
what was Fred Sanger’s Dna sequencing approach (1)
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 .
what was Fred Sanger’s Dna sequencing approach (2)
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 .
what was Fred Sanger’s Dna sequencing approach (3)
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 .
what was Fred Sanger’s Dna sequencing approach - how was the dna fragments read
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 .
how gould was Fred Sanegrs DNA sequencing approach and why
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
How to clone DNA (1)
the gene to be sequenced is siolted using RESTRICTION ENZYMES from a bacterium .
How to clone DNA (2)
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 .
How to clone DNA (3)
Each new bacterium contained a copy of the candidate gene . These lengths of NDA were isolated using plasmid perparation techniques and were then sequenced .
What was the first DNA sequencing machine
-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 .
What was the first DNA sequencing machine (2)
-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 .
What is high throughput sequencing
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
When ws pyrosequencing developed and what is the overall process
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 .
stage one of pyrosequencing
a long length of DNA to be sequences is mechanically cu into fragments of 300-800 base pairs using a nebuliser .
stage two of pyrosequencing
these lengths are then degraded into single stranded dna (ssDNA) . These are the template DNAS nd they are immbollised .
stage three ofpyrosequencing
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 .
stage four of pyrosequencing 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 .
stage four of pyrosequencing b
as this happens the two extr phosphorl are released as pyrophosphate . (ppi)
stage four of pyrosequencing s c
in the presence of AP , the enzyme ATP sulfurylase , converts the pyrophosphate to ATP .
stage five of pyrosequencing d
in the presenc eof this ATP , the enzyme luciferase converts luciferin to oxylucifierin .
stage six pyrosequencing e
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 .
stage seven of pyrosequencing
unincorporated activated nucleoitde are degraded by apyrase and the reactions starts again with another nucleotide .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
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 .
aspects of syntheitc biology
biofules
-biomedicine
-chemistry
-computer science
-engineering science
-physics .
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
applications of syntehtic biology - productions of medicines
escherial coli nd yeas egenticial engineered to produce antimalrial durg .
applications of annaotechnolgoy - nanotechnology
materials can be produced for nanotechnology e.g amyloid fibred to making biofils ofr function uscha s adheison .
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 .
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 .
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 .
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 .
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 .
stage three of dna profiling
the fragments are separated by gel eelectroophorwesis and stained . Larger fragments travel the shortest distance in the gel .
stage four of dna profiling
a banding pattern can be seen .
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 .
stage six of dna profiling
the banding patterns of the DNA samples can then be compared .
what is the first method invovleing restriction fragment length - it is no longer used
polymorphism - method is laborious so therefore no longer used
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 .
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 .
