Manipulating genomes Flashcards
PCR
polymerase chain reaction
Amplifies short lengths of dna into millions of copies
Step 1 PCR
DNA fragments, primers and free nucleotides join, DNA polymerase
Added to thermocycler
94-96 degrees Celsius causes DNA strands to separate (denature) hydrogen bonds
Step 2 PCR
primers join to their complementary bases (55-68 degrees Celsius)
Provides a starting point for DNA polymerase
Step 3 PCR
temperature increased back up to 72 degrees Celsius (optimum temp for DNA polymerase)
cycle restarts & DNA amplified
Double each time
Applications of PCR
-detecting mutations
-research
-forensic science
-identify viral infections
-monitoring spread of infectious disease
-detection of oncongenes
Method to get DNA fragment
some sections of DNA are palindromic
-restriction enzymes recognise specific palindromic sequences
-they cut the DNA at these places because shape of sequence is complimentary to active site
Sticky ends
small tails of unpaired bases at each end of fragment
Used to join DNA fragment to another piece of DNA that has sticky ends with complementary bases
Gel electrophoresis steps
Extraction of DNA from sample
& fragments made by restriction enzyme
Separation using electrophoresis (small fragments travel furthest)
These bands are colourless but are revealed using radioactive or fluorescent probes
(METHOD TO SEPARATE DNA)
DNA ladder
Tool used to compare base length sequences against a known sequence length
Estimation of size
SDS
charge detergent used to equalise surface charge
So that it moves on molecular mass not charge
Uses of DNA profiling
Aid diagnosis of sickle cell
Guilty/non guilty if a crime
Find father of child
embryo screening
DNA probes
small fragment of nucleic acid that is labelled with an enzyme, radioactive tag or fluorescent tag
probe binds to complementary bases
-locates specific gene for genetic engineering
-identify same gene in a variety of different genomes from different species
genetic counselling
family history of genetic disease
Genetic engineering
genes inserted from one organism into another
recombinant DNA
a composite DNA molecule created in vitro joining foreign DNA
GE step 1
reverse transcriptase will form single stranded complementary DNA (cDNA)
addition of primers & DNA polymerase
makes cDNA into double stranded DNA
whose base sequences code for original desired protein
GE step 2
Plasmids obtained from organisms such as bacteria & mixed with restriction enzymes cut at specific sites
-the cut plasmid has exposed sticky ends
DNA ligase will anneal the free nucleotides to sticky ends
GE step 3
Heat shock treatment
-if bacteria are subjected to periods of hot and cold in presence of calcium chloride
-membranes become more porous and allows in recombinant DNA
Electroporation
high voltage pulse is applied to cell to disrupt the membrane
Electrofusion
electrical fields help introduce DNA into cells
heals with DNA inside cell
Agrobacterium Tumefaciens
infects plants
DNA profiling
procedure
-extract DNA from sample
-Digest sample ( DNA cut into fragments) by restriction enzymes
-separate DNA fragments using electrophoresis
-Bonding pattern produced
patterns compared
tandem repeat sequences
-non coding regions
-10-100 base pairs long
-all feature same core sequence
-introns/ telemeres satelite DNA
-similar tandem repeat sequences in relatives
DNA sequencing
Identifying the base sequence of a DNA fragment
Fred Sangers
use a single strand of DNA as template for 4 experiments in separate dishes
Each dish contains solution made up of 4 bases (ATGC) , DNA polymerase
Each dish has a modified version of one of the DNA bases
(modification = being labelled with a radioactive isotope)
terminators
the modified nucleotides
mixture in reaction
-The DNA being sequenced
-mixture of (normal) nucleotides
-one type of terminator nucleotide (modified)
- a primer
-DNA polymerase
Genome sequencing
-genome cut into smaller fragments (restriction enzymes)
-fragments inserted into BACs
-BACs inserted into bacteria
-Bacteria then divide
(creating colonies of cloned cell)
complete genomic DNA library
-DNA then extracted from each colony & cut up using RE
DNA fragments then put back in order by computers
Automated sequencing
-Nucleotides are fluorescently labelled with dyes
-Everything occurs in single tube
separation can occur in one lane during gel electrophoresis
Pyrosequencing
Involves synthesising ssDNA
strand sequenced one base at a time and using light emission which base was added at each time
Pyrosequencing steps
long length of DNA to be sequenced is mechanically cut into fragments of 300-800bp using nebuliser
- these lengths are often degraded into single stranded DNA (ssDNA)
-these are template DNAs and are immobilised
-Sequencing primer added & DNA is incubated with the (DNA polymerase, ATP sulfurylase, luciferase, apyrase
& adenosine 5 phosphosufate
Activated nucleotide
(nucleotide with 2 extra phosphoryl groups)
eg TTP incorporated into complementary strand of DNA by using strand as template
As this happens the 2 extra phosphoryls are released pyrophosphate
ATP sulfurylase
in presence of APS
converts pyrophosphate –> ATP
luciferase
converts luciferin –> oxyluciferin
generates visible light
Bioinformatics
study of sequences
Benefits of comparing genomes
Allow us to compare between species to see evolutionary relationships
Comparing between individuals allows us to tailor medical treatments
Process of inserting DNA fragment into a vector
Plasmid used as vector & is cut using the same restriction enzymes so that ends are complimentary
-DNA ligase joins fragment and plasmid together
Process of inserting vector into host cell
host cells mixed with vectors in ice cold solution then shocked to increase permeability of cell membrane (electroporation) which encourages the cells to take up the vector
disadvantages of genetic engineering
GE seeds would be harder to aquire for poor farmers
GM crops may cause antibiotic resistance
advantages of genetic engineering
GM animals used to make pharmaceuticals
GM foods have longer shelf life
GM food can have better nutrient content
DNA sequencing
Knowing the sequence of a gene allow us to predict the sequence of amino acid that makes up the polypeptide
applications of gene sequencing
comparison between species
evolutionary relationships
variation between individuals
synthetic biology
science concerned with designing and building useful biological devices and systems.
