Manipulating Genomes Flashcards
Genome
Entire genetic material of an organism - DNA in the nucleus and the mitochondria combined
How is DNA layout?
Chromosomes made up of hundreds of millions of DNA base pairs - genes only make up about 2% of your total DNA - these are exons
What are introns?
Large non-coding regions of DNA that are removed from messenger RNA before it is translated into a polypeptide chain
DNA profiling?
Producing an image of the patterns in the DNA of an individual - assists in the identification of individuals/family relationships
What is DNA profiling referred to as?
Genetic fingerprinting since every individual’s DNA profile is unique
5 main stages in producing DNA Profile
Extracting the DNA
Digesting the sample
Separating the DNA fragments
Hybridisation
Seeing the evidence
Extracting the DNA
Using Polymerase Chain Reaction - tiniest fragment of tissue can give scientists enough DNA to develop a profile
Digesting the sample
Strands of DNA are cut into small fragments using special enzymes called restriction endonucleases - different enzymes cut DNA at a specific nucleotide sequence known as a restriction site
How many cuts do restriction endonucleases make?
Two - one through each strand of DNA
Why do scientists use restriction endonucleases?
Give scientists ability to cut the DNA strands at defined points in the introns ; use a mixture of restriction enzymes that leave the repeating units or satellites intact so the fragments at the end of process include a mixture of intact mini and micro satellite regions
Separating the DNA fragments?
Using electrophoresis - the way charged particles move through a gel medium under the influence of an electric current - gel is immersed in alkali in order to separate the DNA double strands into single strands - single stranded DNA fragments are then transferred onto a membrane by southern blotting
Hybridisation
Radioactive or fluorescent DNA probes are now added in excess to DNA fragments on the membrane ; these are short DNA/RNA sequences complementary to a known DNA sequence which bind to complementary strands under particular conditions of pH and temperature
What does hybridisation do?
Identifying micro satellite regions that are more varied than larger mini satellite regions
Seeing the evidence
If radioactive labels were added to the DNA probes - X-ray images could be taken of the paper/membrane ; or fluorescent tags under UV light - fragments give a pattern of bars which is unique except for identical siblings
What is PCR used for?
Amplifying a DNA sample
Ingredients for PCR
Excess of the 4 nucleotide bases (in the form of deoxynucleoside triphosphates)
Small primer DNA sequences
Enzyme DNA polymerase
ALL IN A THERMAL CYCLED
Step 1 of PCR
Temperature is increase to 90/95 which denatures the DNA by breaking hydrogen bonds holding them together so they separate
Step 2 PCR
Annealing of the primers - temperature is decreased to 55/60 and the primers bind (anneal) to the ends of the DNA strands - they are needed for the replication of the strands to occur
Synthesis of DNA PCR
Step 3
Step 3 PCR
Temp is increased to 75 for optimum temperature for DNA polymerase
DNA polymerase adds bases to the primer building up complementary strands of DNA and so producing double stranded DNA identical to the original sequence
What enzyme is used in synthesis of DNA?
Taq polymerase - obtained from thermophilic bacteria found in hot springs
Uses of DNA profiling
Forensics - dna profiles from blood, semen etc is compared to a criminal DNA database
Prove paternity of a child - demonstrates evolutionary relationships between different species
Identifying individuals who are more at risk from developing certain diseases - certain non-coding micro satellites have been found to be associated with increased incidence of disease
DNA profiling summary
Used with DNA sequencing to make more confident risk assessments
Producing an image of the patterns in non-coding DNA of an individual
DNA sequencing
Determine the precise order of nucleotides within a DNA molecule
First sequencing technique?
Sanger sequencing enabled him to read 500-800 bases at one time - first genome they sequenced was phiX174 - virus that attacks bacteria
Refinement of DNA sequencing technique?
Swapping of radioactive labels for coloured fluorescent tags which led to scaling up and automation of the process - led to capillary sequencing version which is still used today
HGP
Massive international project established in 1990 to map the entire human genome - making the data freely available to scientists all over the world
Why was human genome first draft complete so quickly
Automation of sequencing techniques and development of faster computers meant that project was ready two years ahead of schedule and under budget
What are terminator bases?
Modified versions of the 4 nucleotide bases which stop DNA synthesis when they are included - A terminator will stop DNA synthesis at the location that an A base would be added etc etc ; the terminator bases are also given coloured fluorescent tags
Terminator bases lack an oxygen on third carbon
A
Green
G
Yellow
T
Red
C
Blue
What does capillary method start with?
DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases
Step 2 dna sequencing
Mixture is placed in thermal cycles and rapidly changes temperature - 96 degrees would cause double stranded DNA to separate and at 50 degrees the primers anneal to the DNA strand
Step 3 dna sequencing
At 60 degrees, DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template
Step 4 dna sequencing
Each time a terminator base is incorporated - synthesis of DNA is terminated ; as chain terminating based are present in lower amounts and are added at random, it results in many DNA fragments at random lengths - after many cycles, all the possible DNA chains will be produced with the reaction stopped at every base ; they are separated according to length by capillary sequencing (fluorescent markers are used to identify the final base on each fragment) - lasers detect different colours and thus order of the sequence
Order of bases in capillary tubes
Shows the sequence of the new complementary strand of DNA = used to build up original strand ; data fed into computer that reassembles the genomes by comparing fragments and finding the areas of overlap between them - these genomes can then be researched
Next-generation sequencing
DNA sequencing technologies have become faster and more automated - new, automated, high-throughput sequencing processes - instead of using gel of capillaries, the sequencing reaction takes place on a plastic slide known as a flow cell
How does a flow cell work?
Millions of fragments of DNA are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments - as all of the clusters are being sequenced and imaged at the same time, technique is known as ‘massively parallel sequencing’
How refined are these DNA sequencing processes?
Extremely efficient - 3 billion base pairs of human genome can be sequenced in days and those of a bacterium in less than 24 hours ; high-throughput sequencing also means cost has fallen so more genomes can be sequenced and they open up a range of questions that scientists can ask on the genome in many new ways
Bioinformatics
Development if software and computing tools needed to organise and analyse raw data - stats tests, mathematical models and algorithms etc (make sense of a lot of data)
Computational biology
Build theoretical models of biological systems - 3D structures of proteins from sequencing
Determine evolutionary links/genes linked to diseases
Genomics
Analysing structure and function of genomes
Analysing pathogen genomes
Find sources of an infection
Identify antibiotic resistant strains of bacteria
Track of diseases like Ebola
Identify genetic markers in pathogens for use in vaccines
Species genome analysis
DNA barcoding - common sections that vary between species
Mitochondrial dna from cytochrome c - cellular respiration enzyme
Have nit come up with regions for fungi and bacteria (downside)
Phylogeny
Basic mutation rate - can be calculated - how long ago two species diverged
Proteomics
Amino acid sequencing
Spliceosomes
Exons joined together by enzyme complexes called spliceosomes ; in a variety of ways so many proteins from one mRNA
Synthetic biology
Ability to sequence genomes and understand how it translates to amino acids - store and manipulate data
Synthetic biology examples
Genetic engineering
Fixed/immobilised enzymes
New genes replacing faulty genes - development of cystic fibrosis (functional genes replace faulty ones)
Synthesis of entire new organism (artificial genome)
Transgenic
Organism that carries a gene from another organism
Isolate desired gene
Restriction endonucleases cut gene from DNA at specific base sequence
Some are blunt
Others are sticky - one strand longer than other strand ; unpaired/exposed bases makes it easier to insert desired gene into DNA of another organism
How else to isolate gene?
Using reverse transcriptase to produce a single strand of complementary dna - makes it easier to identify the desired gene as some cells make a lot of mRNA
Vectors
Bacterial plasmids - contain marker gene so for example they may have been engineered to have a gene for antibiotic resistance
Bacteria have taken up the plasmid by growing in media containing antibiotic - bacteria will still grow
How to cut open plasmid
Same restriction endonuclease - plasmid has complementary sticky ends - DNA ligase phosphodiester bonds
If DNA fragment inserted successfully
Marker gene will not function - often fluorescence or colour change ; HAS BEEN ENGINEERED SUCCESSFULLY IF IT DOES NOT FLUORESCE
Transferring the vector
Plasmid with recombinant DNA is transferred to host cell in TRANSFORMATION
First way of transformation
Culture in a calcium rich solution and increase temperature - membrane is permeable and plasmids can enter
Second way of transformation
Electroporation small electric current is applied to the bacteria - makes them porous and plasmids move in
POWER HAS TO BE CONTROLLED else membrane will be permanently damaged
Electro fusion
Tiny electric currents are applied to membranes of two different cells - fuses cells and nuclear membranes together to form a polyploid cell continaining both DNA
Used to create a hybridoma
How many different restriction endonuclease needed
2 for each cut
Recombinant plasmid
Genes from organism + marker genes - ONLY FOR DNA
Transgenic is for organism
Restriction endonucleases
At restriction sites
Reverse transcriptase
Allow mRNA to be transcribed - extracted and mix with RE
Converts mRNA back into COMPLEMENTARY DNA AND PUT INTO PLASMID
Useful because it’s mature MRNA (already spliced) SAVES A LOT OF TIME - DO NOT NEED TO PROCESS INTRONS IN PLASMID - SAVES ENERGY FROM BACTERIA
Where do restriction endonucleases cut in vector
At marker gene
Blue marker gene recombinant - DOES NOT PRODUCE BLUE PIGMENT BECAUSE CANNOT BE TRANSCRIBED
Second marker gene - antibiotic resistant gene (can survive with antibiotics)
What enzyme joins fragments together to make recombinant DNA?
DNA ligase
Plasmids only transformed into
Bacteriaaaaa
Blue colonies or
White colonies
Somatic cell gene therapy
Replacing mutant allele with a healthy allele in the affected somatic body cells - a temporary solution as healthy allele is passed on every time a cell divides by mitosis but somatic cells only have limited lives so replaced by stem cells with faulty allele
STILL PASSES ON FAULTY ALLELE TO ANY CHILDREN
Germ line cell gene therapy
Healthy allele into the germ cells of an embryo after fertilisation - individual born healthy and pass onto offspring
Downside of her line cell gene therapy
Potential impact on an individual is unknown
Human rights of unborn individual is violated without consent and irrevocable
Enables people to choose desirable or cosmetic characteristics ETHICAL concerns
Pharming
Production of human medicines - creating animal models or human proteins
Animal models
Genes deleted so they are more likely to develop cancer - develops certain diseases
Human proteins
Human gene coding for a medically required protein - genetic material of a fertilised cow - fertilised transgenic female is then returned to mother ; when baby matures and gives birth produces milk with proteins to be harvested
Ethical issues
Welfare of animals (in pharming for example)
Human genes in animals
Making animals commodities
Insect resistance
Inserted gene into soya beans to produce bt protein that is toxic to many pests - no need for pesticides (or weed killer sometimes)
Benefits of GM crops
Increase yield - no pesticide spraying helps environment
Resistant to diseases
Herbicides can reduce competition
Enhanced levels of vitamins and grown in wider conditions all year round
Produce human vaccines
GM crops cons
Non-pest insects may be damaged by toxins in GM plants
May produce supersedes ; transferred genes in wild populations - reduces biodiversity
People may be allergic
Reduce commercial value and demand - LONG SHELF LIFE
Patent
Legal patent means less economically developed countries cannot use them - flood/drought resistant crops unable to afford the GM seed
CANNOT SAVE THE SEED FOR GROWTH THE NEXT YEAR
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