Please enable JavaScript.
Coggle requires JavaScript to display documents.
biology module 6.3 - manipulating genomes - Coggle Diagram
biology module 6.3 - manipulating genomes
dna sequencing
the process of determining the exact order of nucleotides in a dna molecule
Sanger sequencing :
what is in the test tube ?
DNA (for sequencing)
primer
DNA polymerase
excess nucleotides
terminator base (tagged)
primer = small chain of dna, provides a starting point for DNA polymerase
terminator base = modified base that stops dna synthesis, either CGAT
A single strand of DNA is used as a template, added to 4 separate test tubes (each have a different terminator base in them). DNA polymerase attaches and completes dna replication using the excess nucleotides - until a terminator base is added.
Different lengthen fragments are produced as terminator base will bind and stop dna replication at different points
interpreting results from Sanger sequence :
gel electrophoresis is used
the for test tubes are placed in 4 different wells the top of ager well, electric current is applied, causing -tive charged dna to move towards positive electrode
the shorter dna fragments move faster and reach the bottom quicker
read results from the bottom us
read the tagged terminator bases (write out the complementary bases to determine the actual bases on the dna strand)
pyrosequencing = high-throughput sequencing method where light emissions are emitted allowing us ti determine dna base order
pcr
a technique used to amplify a very small sample of dna to create a sample large enough to be amplified - doubled each time pcr is completed
uses of pcr = paternity test and in forensics
what is needed for pcr ?
dna fragments (sample)
dna polymerase
primer
excess nucleotides
in pcr, a bacteria from hot springs (Taq) is used as DNA polymerase because it can withstand the hot temperatures - thermostable
stage one =
denaturation
split dna into 2 separate strands
happens at 95 degrees
stage two =
annealing
2 primers are added to the separate dna strands
happens at 65 degrees
temp drops to help primers bind to dna strands
stage three =
extending
2 dna polymerase molecules attach and create complementary dna strands (double the dna)
happens at 72 degrees
uses of dna sequencing
bioinformatics = development of software to organise and analyse raw dna data - used to workout source of outbreaks and find vulnerable populations
computational biology = using biological data from bioinformatics to create models of biological systems
genome wide comparison = determine the complete DNA sequence of an organism
help us to find evolutionary links between species
compare induviduals of the same species
human - genome project = aim to determine the order of base pairs in a human, identify all genes and sequence these genes and map them out on a chromosome
genome wide comparison on pathogens :
doctors can find source of infection, track the spread of disease, identify antibiotic resistant strains of bacteria, create drugs
synthetic biology
knowing the sequence of a gene allows us to predict the aa sequence because we know the triplet code that codes for the aa, we can predict the primary structure of the protein allowing biological molecules to be created from scratch
dna profiling
dna profiling uses small fragments of dna sample to determine whether they come from the same individual
uses of dna profiling :
forensics
disease analysis
parental disputes
satelite dna = short sequence of dna that is repeated many times, found in introns (section of dna that doesn't code for a protein)
minisatelite = segment of repeating dna (20-50 bases repeated hundreds of times), VNTR
microsatelite = segment of repeating dna (2-4 bases repeated 15 times), STR
number of satellite repeats matches that of the other person = more closely related
process of dna profiling :
1) extract and purify the DNA :
use pcr to amplify dna
add protease to dissociate histones from dna
2) digest DNA with restriction enzyme
use restriction endonuclease to cut out STRs at recognition sites
3) DNA fragments are different lengths
use electrophoresis to separate the fragments
submerge the double stranded dna in alkali solution to make it single stranded
4) transfer single DNA strands onto membrane
process = southern blotting
5) Hybridisation
dna probes added in excess to the single dna fragments on the membrane
probes attach to complementary fragments
dna probes identify microsatelite regions and attach to them (help identify them)
6) viewing the STRs
if the dna probe was a radioactive tag, use x-ray to view
if the dna probe was a fluorescent tag, use uv light
dna probe = a tagged single stranded nucleic acid molecule (made with complementary bases to the gene you want to locate)
genetic engineering
summary of stages :
1) obtain and isolate the gene
2) place gene inside vector
3) vector carries gene into recipient cell
4) recipient cell expresses the gene
obtain the gene
locate the gene using DNA probes with tags
extract mRNA from a cell transcribing the gene
extract using restriction endonuclease
convert mRNA into cDNA using reverse transcriptase
DNA polymerase convert cDNA to double stranded DNA
amplify DNA using pcr (if needed)
place gene inside vector
cut the plasmid inside the vector using restriction endonuclease
insert gene into plasmid
DNA ligase seals the Okazaki fragments together
vector carries gene into recipient cell
electroporation = help dna cross the membrane by applying a voltage that creates punctured holes
insulin via replica plating
original plasmids have antibiotic resistance gene to antibiotics ampicillin and tetracycline
cut out tetracycline resistance gene and insert insulin gene
= no more resistance to tetracycline
bacteria are grown on agar plates
transferred onto plate ctreated with ampicillin
= to see if the gene has been take up or not
transferred onto a plate treated with tetracycline
no growth = contain insulin gene
gene therapy
germ line therapy = inserting a healthy allele into the germ cells (cell that develops into a reproductive cell) / very early embryo
corrected gene inserted into embryo via IVF, all cells of the embryo will contain corrected gene
permanent method, offspring inherit the corrected gene
somatic cell therapy = replacing a faulty gene with a healthy allele in affected somatic cell (any cell other than reproductive cell)
copies of the corrected gene are directly inserted into the body cell of sufferer
does not prevent it being passed onto offspring as it doesn't affect sperm/egg cells and it has to be repeated many times + doesn't last long