Manipulating Genomes Flashcards
(30 cards)
What is genetic fingerprinting?
DNA profiling
A technique used to identify unique DNA patterns in individuals to help identify individuals in forensics or family relationships
What are variable number tandem repeats?
Variation in sequence NS length of unique non coding, repetitive DNA segments
What are features of variable number tandem repeats?
- present across the genomes of most eukaryotes
- not involved in protein coding
- extensive variability in sequence and length
- ## length and location are variable
What are short tandem repeats?
Repeated sequences of nucleotides that are smaller than VNTS and can be used for genetic fingerprinting
Describe the process of creating a genetic fingerprint:
- DNA extraction - DNA is extracted from a tissue sample and amplified using PCR
- DNA digestion - restriction enzymes are used to cut the DNA fragments at points near the VNTR sequences
- Fragment separation - gel electrophoresis separates the fragments by size and they denatures to produce singular strands
- Hybridisation - specific radioactive or fluorescent probes bind to complementary VNTR sequences.
- Development - the positions of the probes are revealed, creating a barcode - like pattern of DNA bands unique to each individual
What is a genome, what is a proteome?
How can this be used?
Genome is all the genetic material an organism contains
A proteome is all the proteins a cell can produce
By knowing the genome sequence, the proteome can ve derived from genetic code which can have applications including identification of possible antigens fo ruse in vaccination productions.
Howver in more complex organisms the presence of non coding and regulatory genes means that knowledge of genomes isn’t easily translated into proteome
Sequencing methods
… continuously being updated with new research which has increased the speed of sequencing and allowed the whole genome to be sequenced through different method
Processes have become faster and automated compared to original Sanger method but many principles remain the same
- terminator bases that stop DNA synthesis at random pints
- DNA polymerase, primer, excess of nucleotides and terminator bases and the DNA sequence is mixed together in PCR to amply DNA fragment
- continues until terminator bases have all been made
- terminator bases are labelled with different fluorescent colours so they can be ideniftied
- DNA fragments are separated using gel electrophoresis according to length/size
- used to work out exact sequence of bases
High input frequenting is when many fragments are processed and sequenced simultaneously to make the process more efficient
What has gene sequencing made it possible for scientist to do?
Compare entire genomes of organisms of the same of different species
Analyse pathogen genomes resulting in:
- identifying a source of an infection
- identifimg antibiotic resistant bacteria
- tracking teh spread of pathogens to monitor potential pandemic a
- identifying regions in the genome for new drugs to target
Comparing genomes has:
- improved the accuracy of classification of species
- understand evolutionary relationships, similar sequences can suggets how recently they evolved from a common ancestor
Why cant we predict proteomics in humans?
The human genome contains thousands of genes and predictions could vary into millions
This has still taught us that the re;ationship between genotype and phenotype is more complex than originally thought
Why is synthetic biology, how has this been enabled?
Synthetic biology is teh creation of artificial pathways, organisms or systems of natural systems
This has been enabled by sequencing genomes
E.g
Genetic engineering (insulin producing bacteria)
Synthesis of new genes to replace faulty versions of genes
What is bioinformatics and computational biology?
Bioinformatics - use of software to analyse, organise and store biological data. Includes databases storing all known alleles, amino acids sequences and protein structures
Computational biology - using computers to study biology e.g simulations, models, algorithms - protein structures can be modelled and effects of mutations observed
What is STR?
What is their relavance in genetic fingerprinting?
Short tandem repeats
95% of human DNA is made up of introns which are non coding DNA sequences. They consist of STR.
The proabanlity of two individuals having teh same STRs is very low but the more closely related teh more similar the STRs are
Analysing STRs in genetic fingerprinting allows to determine how closely related two individuals are.
Process of genetic fingerprinting:
- Collection of DMA sample
- PCR of DNA to amplify it so can be repeated
- Digestion - cutting DNA into smaller fragments using restriction enzymes which cut at specific recognition sites close to STRs
- Separate DNA fragments using gel electrophoresis: small wells in agar gel, DNA is negatively charged to moves towards positive anode, separates by length
- An alkaline is added so DNA sections are split into single strands breaking hydrogen bonds so ….
- Hybridise single strands with DNA probes that are complimentary to STRs, align and hybridise
- Needed so we can see where bands of DNA are becasue DNA probes have fluorescent label
- Once probes bind, rinse gel and transfer onto nylon sheet
- Use an UV light to view fluoresce probes, making DNA bands visible
DNA bands can be compared to ideniftied a genetic relationship between known ands unknown samples
Protein electrophoresis
Used in diagnosis of medical conditions where an abnormal protein is responsible for the disease.
Method is the same but they must be denatured first so they can pass through gel, this is done by heating the proteins.
As they can be charged, they need to be made to have a negative charge using chemicals
Advantages of PCR
Automated, rapid, doesnt require living cells like invivo cloning does
In vivo cloning
- Restriction enzymes cut DNA fragments
- DNA needs to be inserted to vector which carries isolated DNA fragments into hos t(bacteria)
- Plasmids are most common
Type of vector depends on the organism that is being genetically modified, prokaryotes use a plasmid of bacteriophage. In eukaryotes a virus of liposome can be used
- The plasmid is cut open using the same restriction enzyme used to cut dna fragment which makes sticky ends in the plasmid that will be complimentary to sticky ends on DNA fragment
- DNA fragments and plasmid are combined and enzyme ligase is used to bind them and catalyse formation of phosphodiester bonds between nucleotides to create recombinant plasmid
Transformation: getting plasmid into host cell ( bacterium) where gene will be expressed to create protein
- Cell membrane must be made more permeable by mixing host cells with calcium and heat shock, electron oration is used so an electrical current is applied to the membrane to makeit more porous
- This enables vector to open host cells cytoplasm
1.
What are restriction enzymes?
Cut at recognition sites leaving sticky ends, they cut up DNA
They occur naturally in bacteria as a defence mechanism
Many have an active site that is com0limentry in shape to a range of different DNA base sequences called recognition sequenced
Each enzyme cuts at a specific location
Some enzymes cut at the same location in teh double strand which creates a blunt end so there is no overhang
Sticky ends are the same forwards and backward (palindromic) so they have the ability to join DNA with complimentary base pairing
How can you identity which cells have been transformed in in vivo cloning?
Use off gene markers
Why would a host cell not take up the recombinant plasmid?
The plasmid doenst get insid ethe cell, the plasmid rejoined before DNA fragment enters, the DNA fragment ticks to itself rather than plasmid
How are marker genes used?
They are used to idenifty whether a plasmid vector has entered a host cell or not
Why might the vector not enter the host cell in in vitro cloning?
The DNA fragments sticky ends connected to itself instead of plasmid
The plasmid did not eneter host cells with calcium becsue membrane wasnt permaeble enough
How to marker genes work?
DNA fragment is interest to the gene that is a marker gene. Thsi disrupts it and stops teh gene from working.
Antibiotic resistant genes are often used as marke genes
They can be identified by growing all the bacteria on an agar place, use a sterile block to take an imprint of all teh colonies, and print them onto new Petri dish which has agar and an antibiotic in the agar.
See which colonies are able to grow, these loonies were resistant to the antibiotic which means their antibiotic resistant gene is functional so they are not carrying teh DNA fragment
Genetic engineering in plants:
DNA of crops have had genes added to make them pest resistant, disease resistant and herbicide resistant resulting in higher yields
DNA manipulated to have a longer shelf life or to have more nutritional value to produce medicine
Perceived negatives:
Genes could spread to other plants in the environemnt
Concerns that people may be allergic to different proteins that some crops may now make
Technology is often patented so buying genetically engineered seeds is expensive and only affordable to some farmers, no poorer farmers
