6.1.3 Manipulating Genomes Flashcards
What is a genome and what does it consist of?
-all of the genetic material within an organism
-made up of DNA in the nucleus contained within chromosomes and mitochondria
Explain what short tandem repeats are
-exist within introns, telomeres and centromeres
-short sequences of DNA that are repeated many times–} known as microsatellites made up of repeated regions of 2-4 bases
-always appear on the same position on the chromosomes but the number of repeats vary per individual–} different length repeats inherited from each parent= unique pattern
-more closely related you are, the more likely you are to have similar patterns i.e identical twins(allows for family mapping/paternity testing)
Why do STRs exist in introns and not exons?
-because our exons are highly conserved as they are essential for protein synthesis, whereas intron are not expressed as they are non-coding regions of DNA
What is a DNA profile?
-a unique DNA sequence that allows an individual to be identified based on the STRs at each locus on their chromosomes
What are the steps of making a DNA profile?
-DNA extraction
-Amplify DNA using PCR
-Cut up DNA fragments using restriction enzymes
-Separate DNA using gel electrophoresis
-Visualisation through hybridisation
-Comparison of DNA samples or sequencing
DNA extraction
-break up the sample using a pestle and mortar to break up the cell wall
-make up a solution of detergent(breaks up cell membrane and nuclear envelope), salt(clumps up the DNA) and distilled water and add the cells to the solution beaker
-incubate the beaker at 60 degrees Celsius (denatures digestive enzymes)
-put beaker into an ice bath to cool the mixture down
-filter the mixture to isolate DNA extract
-add protease to break down histones and expose the DNA
-slowly dribble cold ethanol down the side of the test tube(causes DNA to precipitate as it is insoluble in alcohol)–} can be extracted with a glass rod
Define all the substances needed for PCR
-Taq DNA polymerase= enzyme found in bacteria from thermal hot springs–} can withstand high temps and doesn’t denature at 95°(many cycles of PCR can be carried out without needing new enzymes each time), used to create new DNA strands
-Primers= short, single-stranded pieces of DNA complimentary to the start of the sequence to be amplified
Describe how DNA is amplified using PCR
-PCR is used to create many copies of DNA from a fragment
- 95°C:
-DNA mixture is heated to 95° for 30 seconds–} breaks the hydrogen bonds and separates the 2 strands of DNA
- 55°C:
-DNA mixture is cooled
-complimentary primers anneal to the end of each strand
- 72°C:
-DNA mixture is heated for optimum conditions for Taq to speed up the reaction
-Taq DNA polymerase lines up free nucleotides onto the primer complimentary the template strand
What are the results of one PCR cycle?
-2 new double stranded fragments of DNA identical to the original sample
(the amount of DNA doubles each cycle i.e 16 cycles= 2 to the power of 16)
Explain what restriction enzymes are?
-some sections of DNA have palindromic sequences of nucleotides(consist of antiparallel base pairs)
-restriction enzymes will recognise and cut the DNA at the restriction site
-the active site of a restriction enzyme is specific to the complimentary shape of the restriction sites
How do restriction enzymes work?
-the DNA sample is incubated with the specific restriction enzyme
-hydrolysis reaction occurs
breaking sugar phosphate backbone of DNA via breaking down phosphodiester bonds using water
-reaction often results in a ‘staggered cut’ leaving exposed bases at the end of each fragment called sticky ends –} can be used to bind the DNA fragment to any other DNA fragment that has complimentary sequence at the sticky ends
Separating DNA using gel electrophoresis
-uses an electrical current to separate out DNA fragments
-gel tray is added to gel box/tank, the end with wells is positioned next to the cathode(negative)
-gel tray is immersed in a buffer solution(keeps reaction alkaline which maintains the pH and helps carry the current)
-a set vol of loading dye is mixed with each sample to be separated(ensures DNA sink to the bottom of the wells and helps visualise the DNA)
-ladder(DNA sample of known fragment lengths) is loaded into the first well as a control
-gel box is closed and connected to power supply(cause current to run through gel)
-negative DNA fragments move through mesh like structure of the gel–} rate of movement depends on mass/length of DNA fragment, shorter=quicker and vice versa
How can electrophoresis be carried out on RNA fragments and proteins?
-follow the same basic DNA method for RNA
-proteins can be negatively OR positively charged, so they are mixed with a chemical(SDS) to coat the protein with a negative charge and denature it so it is at its primary structure
What is southern blotting?
-after electrophoresis, fragments are placed in an alkaline buffer solution which denatures the DNA so it is single-stranded(exposed DNA stands so probes can bind via CBP)
-through southern blotting, strands are transferred to a nylon membrane in exactly the same position relative to the gel–} fixed in place using UV light or heated at 80°C
Visualisation through hybridisation
-probes are short DNA/RNA fragments complimentary to known STRs
-radioactive or fluorescent DNA probes are now added to the DNA fragments on the membrane
-bind to complimentary strands of DNA and identify STRs
How are the different probes visualised?
-If radioactive labels were added to the probes, then x-ray images are taken of the electrophoresis results
-if fluorescent labels were added to the probes, the electrophoresis results are placed under a UV light so the fluorescent tags glow
What are the uses of DNA profiling?
-can be used in forensic science: DNA traces can be obtained from crime scenes and DNA profile can be compared to suspect(has to be a profile that does not match that of the victim’s)
-can be used in paternity testing
-can be used in medical diagnosis: DNA profile can refer to a unique pattern of several alleles, can be used to analyse the risk of genetic disorders
What is DNA sequencing?
-the process of determining the order of nucleotide bases within a DNA molecule–} allows you to find out the amino acid sequence + the polypeptide it codes for
What is needed for chain termination sequencing?
-into each of the 4 thermal cyclers you will add: the single stranded DNA fragment to be sequenced, taq DNA polymerase, DNA primers, free nucleotides and a fluorescently-labelled modified nucleotide(lack hydroxyl group so sequencing cannot continue–} no phosphodiester bonds to continue sugar phosphate backbone, known as terminator bases)
What are the following steps of chain termination sequencing?
-during PCR, each thermal cycler will produce DNA of each known length ending in a modified nucleotide–} strands are different lengths depending on where the modified nucleotide was added
-the fragments are then added to separate wells in gel electrophoresis
-smallest fragment(the base at the start of the sequence) will travel the furthest
-the sequence can be read up from anode to cathode to build the DNA sequence one base at a time
-sequence is complimentary to the original sequence
What is automatic sanger sequencing?
-chain termination PCR ran in one tube (with ddNTPS with fluorescents tags specific to the nitrogenous base)
-the fragments are loaded into a single capillary as opposed to multiple wells on a gel tray–} narrow enough so the DNA will be single file which increases resolution
-laser is shone on the end of the capillary and excites the fluorescently tagged terminator nucleotides- the different colour are detected by a computer and thus the sequence order is produced on a chromatogram
Why is genome sequencing needed?
-chain termination method can only be used for DNA fragments up to 750 bp long
-need to make smaller pieces and put them back in order, to sequence an entire genome
Steps of genome sequencing
-genome is cut into smaller fragments using restriction enzymes
-sequence the smaller pieces
-cut the DNA again with a different restriction enzyme + sequence those pieces
-put this back into the correct order using overlapping fragments
Why were advancements in sequencing needed?
-easier because it is now automated
-faster + more efficient so more genomes can be sequenced
High throughput/next generation sequencing
-faster as most methods often have multiple fragments being sequenced in parallel–} cheaper
-but are still based on the principle of adding fluorescent terminator nucleotides
-takes place on a flow cell(plastic cell) instead of gel or capillaries
-i.e pyrosequencing, nanopore sequencing
What is bioinformatics?
-the development of software and computing tools needed to organise/ analyse raw biological data i.e mathematical models, stat tests
What is computational biology?
-the use of computational techniques to use data to build theoretical models of biological systems
-key in identifying genes linked to specific diseases in populations and determining evolutionary relationships
Explain genome wide comparisons
-uses DNA sequencing + computational biology to analyse the structure and function of genomes
-can be compared between the same species or different species
Epidemiological studies
-study of health + disease within a population—} considers distribution, cause + effect of a disease
-computers can analyse + compare the genomes of many individuals, revealing patterns in the inherited DNA + the diseases they are vulnerable to
-comparisons between genomes of people with/ without a disease can lead to detection of particular mutations which put individuals at greater risk of developing the disease(depends on allele)
Studying genotype-phenotype relationship
-useful to be able to predict an organism’s phenotype by analysing its genotype
-data can be used for treatment as they can predict what health conditions a person with this disorder might face
-i.e marfan syndrome is a genetic disorder caused by a mutation of the FBN1 gene—} position of mutation results in different symptoms of the disorder
Analysing the genomes of pathogens
-NGS has allowed for sequencing of pathogen genomes to become fast and relatively cheap
-doctors can:
• find out the source of an infection
• identify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping prevent the spread of resistance
• can track the progress of epidemic/disease outbreak
• identify regions in the genome of pathogens that may be useful targets in the development for treatment
• can be nano pore technology used in pandemic/epidemic zones
Understanding evolutionary relationships
-all organisms evolved from the same common ancestor—} organisms that are closer related would have diverged more recently so they share more DNA
-enables scientists to build up evolutionary trees with accuracy
-whole genomes of different species can be sequenced + analyse via bioinformatics to tell us how closely related different species are
What is synthetic biology?
-ability to sequence the genome of organisms and identify the amino acids that build up the protein—} can use sequencing info to predict the primary structure of polypeptides
-allows scientists to build synthetic biological molecules
-can build new biological systems or edit existing ones
Uses of synthetic biology
-development of biological molecules: enzymes, pigments(tomatoes, blueberries), bacterial cells
-pharmacogenetics: possible to test whether a drug will work by testing peoples genomes, replace ‘one fits all’ method
-genetic engineering: single change in a
biological pathway or relatively major GMO of an organism
-synthesis of new genes to replace faulty genes
What is genetic engineering + how is it possible?
-form of synthetic bio involving the manipulation of the DNA sequences of an organism
-the genetic code says that DNA is universal(all organisms use the same bases and the same codons code for the same AA in all living things) —} genetic info is transferred between species
-transcription + translation are also universal—} the transferred DNA can be translated within cells of the GMO organisms
What are the basic principles of genetic engineering?
-organisms’ DNA can be altered by combining lengths of nucleotides from different sources(may be from the same or different species)
-the altered DNA with the introduced nucleotides is called recombinant DNA
-organism that carries a gene
from another organism in a different species is transgenic
-any organism that has introduced genetic material is a GMO
Step 1: Isolating the desired gene (method 1)
-DNA prove can be used to locate a gene within the genome
-gene can be cut out using restriction endonuclease (breaks the DNA strands at specific base sequences within the molecule) —} leaves sticky ends (overhangs of the DNA that are complimentary) which make it easier to insert the gene into the DNA of another organism
-amplify DNA using PCR
Step 1: Isolating the desired gene (method 2)
-induce expression of the desired gene (forcing cell to synthesise the protein)
-extract mRNA
-use reverse transcripts add to form single stranded complimentary DNA + amplify using PCR
-cut the ends of cDNA with a restriction endonuclease
(makes it easier to identify the desired gene, as a particular cell will make some very specific types of mRNA i.e insuline mRNA)
What are the different types of vectors that can be used in genetic engineering?
-plasmids: transfer DNA into bacteria or yeast
-viruses(bacteriophages): vesicles with phospholipid bilayer used to transfer DNA into human cells/bacteria)
-liposomes: fuse with cell membranes to transfer DNA into cells
-agrobacterium: bacteria often used to transfer DNA to plants
(once a plasmid gets into a new host cell + combines with the host DNA it is called recombinant DNA)
Step 2: making recombinant DNA
• isolated gene is inserted into a vector(used to transfer DNA into a cell):
- vector DNA is cut open using the same restriction endonuclease that was used to isolate the desired DNA fragment/desired gene —} have complimentary sticky ends
- vector and genes are incubated with DNA ligase which catalyses the formation of phosphodiester bonds between the complimentary sticky ends on the sugar phosphate backbones, joining them together to form recombinant DNA
What are marker genes?
-genes which gives the organism a selectable trait if it takes up the plasmid i.e GFP(green fluorescent protein) or antibiotic resistance
what are the role of marker genes in genetic engineering?
-the plasmids that are chosen as vectors have a special marker gene i.e have a gene for antibiotic resistance —} allows scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in culture containing the antibiotics
-plasmids used as vectors are usually given a second marker gene via genetic engineering which is used to show that the plasmid contains the recombinant
gene
-plasmid is then cut using a restriction enzyme within this marker gene to insert the desired gene —} successful insertion = marker gene not functioning
Why are genes resulting in antibiotic resistance being used less frequently as marker genes?
-concerns about antibiotic resistance in GMO as it may lead to widespread resistance
-genes producing fluorescence or an enzyme that causes a colour change in a particular medium are now more widely used
Step 3: Transferring the vector
-vector with the recombinant DNA must
be transferred into the host cell in process called transformation
