6.1.3 Manipulating Genomes

Question 1

What is a genome and what does it consist of?

Answer 1

-all of the genetic material within an organism
-made up of DNA in the nucleus contained within chromosomes and mitochondria

Question 2

Explain what short tandem repeats are

Answer 2

-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)

Question 3

Why do STRs exist in introns and not exons?

Answer 3

-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

Question 4

What is a DNA profile?

Answer 4

-a unique DNA sequence that allows an individual to be identified based on the STRs at each locus on their chromosomes

Question 5

What are the steps of making a DNA profile?

Answer 5

-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

Question 6

DNA extraction

Answer 6

-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

Question 7

Define all the substances needed for PCR

Answer 7

-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

Question 8

Describe how DNA is amplified using PCR

Answer 8

-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 by forming H bonds
- 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

Question 9

What are the results of one PCR cycle?

Answer 9

-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)

Question 10

Explain what restriction enzymes are?

Answer 10

-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

Question 11

How do restriction enzymes work?

Answer 11

-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

Question 12

Separating DNA using gel electrophoresis

Answer 12

-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

Question 13

How can electrophoresis be carried out on RNA fragments and proteins?

Answer 13

-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

Question 14

What is southern blotting?

Answer 14

-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

Question 15

Visualisation through hybridisation

Answer 15

-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

Question 16

How are the different probes visualised?

Answer 16

-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

Question 17

What are the uses of DNA profiling?

Answer 17

-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

Question 18

What is DNA sequencing?

Answer 18

-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

Question 19

What is needed for chain termination sequencing?

Answer 19

-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)

Question 20

What are the following steps of chain termination sequencing?

Answer 20

-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

Question 21

What is automatic sanger sequencing?

Answer 21

-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

Question 22

Why is genome sequencing needed?

Answer 22

-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

Question 23

Steps of genome sequencing

Answer 23

-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

Question 24

Why were advancements in sequencing needed?

Answer 24

-easier because it is now automated
-faster + more efficient so more genomes can be sequenced

Question 25

High throughput/next generation sequencing

Answer 25

-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

Question 26

What is bioinformatics?

Answer 26

-the development of software and computing tools needed to organise/ analyse raw biological data i.e mathematical models, stat tests

Question 27

What is computational biology?

Answer 27

-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

Question 28

Explain genome wide comparisons

Answer 28

-uses DNA sequencing + computational biology to analyse the structure and function of genomes -can be compared between the same species or different species

Question 29

Epidemiological studies

Answer 29

-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)

Question 30

Studying genotype-phenotype relationship

Answer 30

-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

Question 31

Analysing the genomes of pathogens

Answer 31

-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

Question 32

Understanding evolutionary relationships

Answer 32

-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

Question 33

What is synthetic biology?

Answer 33

-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

Question 34

Uses of synthetic biology

Answer 34

-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

Question 35

What is genetic engineering + how is it possible?

Answer 35

-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

Question 36

What are the basic principles of genetic engineering?

Answer 36

-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**

Question 37

Step 1: Isolating the desired gene (method 1)

Answer 37

-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

Question 38

Step 1: Isolating the desired gene (method 2)

Answer 38

-induce expression of the desired gene (forcing cell to synthesise the protein) -extract mRNA -use reverse transcriptase 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)

Question 39

What are the different types of vectors that can be used in genetic engineering?

Answer 39

-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)

Question 40

Step 2: making recombinant DNA

Answer 40

• 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

Question 41

Why is there a low success rate?

Answer 41

-many of the plasmids/vectors end up resealing during incubation

Question 42

What are marker genes?

Answer 42

-genes which gives the organism a selectable trait if it takes up the plasmid i.e GFP(green fluorescent protein) or antibiotic resistance

Question 43

what are the role of marker genes in genetic engineering?

Answer 43

-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 -having 2 or more marker genes reduces the chance of false positives

Question 44

Why are genes resulting in antibiotic resistance being used less frequently as marker genes?

Answer 44

-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

Question 45

Step 3: Transferring the vector

Answer 45

-vector with the recombinant DNA must be transferred into the host cell in process called **transformation** -one method is to culture the bacterial cells + plasmids in a calcium-rich solution and increase the temp--} causes bacterial membrane to become permeable so plasmids can enter -chemical transformation: heat shock bacteria so that it will take in plasmids from environment as it is under stress -2 other methods: electroporation & electrofusion

Question 46

Electroporation

Answer 46

-a small electrical current is applied to the bacteria--} causes membrane to become porous to allow plasmids to move into the cells (only works for plasmid vectors + has low success rate) -can also be used to get DNA fragments directly into eukaryotic cells--} new DNA passes through cell membrane + nuclear membrane to fuse with nuclear DNA

Question 47

Electrofusion

Answer 47

-small electrical currents are applied to the membranes of 2 different cells to fuse the cell + nuclear membranes together to form a hybrid cell containing DNA from both -successfully used to produce GM plants + monoclonal antibodies in animals

Question 48

What happens at the end of the transferring step?

Answer 48

-there are 3 types of bacteria: one with 2 marker genes, one with recombinant DNA and one with no plasmid -to differentiate between these, they can be placed on an antibiotic plate--} one which dies immediately/fails to grow has no plasmid -bacteria that fluoresces doesn't contain the recombinant DNA

Question 49

How is a bacteriophage vector transferred?

Answer 49

-it will infect the host bacterium by injecting its DNA into it -the phage DNA(with the desired gene in it) then integrates into the bacterial DNA

Question 50

GM of microorganisms

Answer 50

-microorganisms(typically bacteria or yeast) can be used to store a living record of DNA of another organism--} source of DNA fragments -pathogens are sometimes GM for medical + epidemiological studies but is strictly regulated -scientists are carrying out research into genetically engineered pathogens i.e viruses in order to find treatment for diseases

Question 51

Ethical issues of using GM microorganisms

Answer 51

-researchers could be infected with pathogen and cause a mass outbreak -GM pathogen could revert back to being pathogenic and cause a mass outbreak -research could be used with malicious intent i.e biowarfare :) GM to produce useful substances i.e insulin + vaccines, strict protocols when using pathogens

Question 52

creating insect-resistant plants

Answer 52

-yields of important food i.e soybeans sources can be reduced by insect pests that feed on them -scientists have inserted a gene into soybeans using agrobacterium as the vector--} leads to production of Bt protein that is toxic to many pest insects

Question 53

GM crops with longer shelf life

Answer 53

-scientists silenced a gene in tomatoes that made them responsible for ripening as an alternative to picking crops when they are unripe and treating them with ethene

Question 54

Advantages of GM crops

Answer 54

-pest resistant: reduce pesticide use, protects environment, helps poor farmers, increases yield -disease resistant: increases yield -herbicide resistant: reduces competing weeds + increases yield -extended shelf life reduced food waste - crops grow in wider range of conditions i.e flood/drought resistant -can be increased in nutritional value i.e golden rice -plants could be used to produce meds/vaccines

Question 55

Disadvantages of GM crops

Answer 55

-toxins in the plant may affect non-pest insects + predators, pests may become resistant -disease resistant genes may spread to wild populations via horizontal gene transfer -biodiversity could be reduced by overuse of herbicides -extended shelf life= reduced commercial value + demand for crop -people may be allergic to different proteins made by GM crops

Question 56

Patenting issues

Answer 56

-major concern is that people in less economically developed countries will be prevented from using GM crops--} restricts those who need drought/flood resistant crops or high nutritional value most from accessing it

Question 57

GM animals: medical research

Answer 57

-scientists can GM animals to have medical conditions--} use them as models to test new medicine -i.e knockout mice whose genes have been manipulated to study diseases like Parkinson's, heart disease etc

Question 58

GM animals: Pharming

Answer 58

-genetically engineering animals to produce pharmaceuticals(via synthetic biology) -done by: creating animal models creating human proteins (human gene introduced to the genetic material of a fertilised animal egg, embryo is then returned to mother + transgenic female is born--} its milk will contain the desired human protein + can be harvested)

Question 59

Ethical issues of GM animals

Answer 59

-religious obligations -could cause harmful side-effects for the animal -welfare compromised :) drugs can be made in large quantities compared to other methods of production

Question 60

Somatic cell gene therapy

Answer 60

-replacing mutant allele with healthy allele in the affected somatic (body) cell :) successful treatments reported i.e immune diseases, leukemia :( problems getting healthy alleles into the affected alleles + starting and maintaining expression of healthy allele, only temporary as somatic cells have a limited life and are replaced from stem cells which will have the faulty gene

Question 61

Germ line cell gene therapy

Answer 61

-inserting a healthy allele into the germ cells (usually the eggs) or into an embryo immediately after fertilisation--} born healthy with normal allele in place which they pass on to their own offspring :) successful with animal embryos :( illegal for human embryos in most countries--} ethical concerns i.e rights of embryos and enabling desirable characteristics, medical concerns i.e impact of germ cell intervention is unknown