Manipulating genomes Flashcards
Fred Sanger DNA sequencing approach
-use a single strand of DNA as template for 4 experiments in separate dishes
-each dish contained a solution with 4 bases A,T,C,G plus enzyme DNA polymerase
-to each dish, modified version of one of DNA bases was added
-base was modified so once incorporated into synthesises complementary strand of DNA, no more bases could be added
-each modified base was labelled with radioactive isotope
-reaction generated thousands of DNA fragments of varying lengths
-DNA fragments were passed through gel by electrophoresis
-smaller fragments travelled further- were sorted by length
-the nucleotide base at end of each fragment was read according to its radioactive label
-the method was efficient and safe however it was time consuming and therefore costly
Cloning DNA
-the gene to be sequenced was isolated using restriction enzymes from a bacterium
-DNA was then inserted into bacterial plasmid (vector) and then into E.coli bacterium host, that, when cultured, divide many times, enabling plasmid with DNA insert to be copied many times
-each new bacterium contained a copy of the candidate genes
-these lengths of DNA were isolated using plasmid preparation techniques and were then sequenced
First DNA sequencing machine
-1896 first automated DNA sequencing machine was developed based of Sangers method
-fluorescent dyes instead of radioactivity were used to label the terminal bases
-the dyes glowed when scanned with laser beam and light signature was identified by computer
-this method dispensed with the need for technicians to read autoradiograms
High throughput sequencing
1) long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs using nebuliser
2) these lengths are then degraded into single stranded DNA (ssDNA)
These are the template DNAs and are immobilised
3) a sequencing primer is added and the DNA is then incubated with enzymes DNA polymerase, ATP sulfurylase, luciferase, APS and luciferin.
Only one of the 4 possible activated nucleotides ATP, TTP, CTP and GTP is added at any one time and an light generated is detected
4) one activated nucleotide (nucleotide with two extra phosphoryl groups) such as TTP is incorporated into complementary strand of DNA using strand to be sequenced as a template
-As this happens two extra phosphoryl are released as pyrophosphate (PPi)
-in the presence of APS the enzyme ATP sulfurylase converts the pyrophosphate to ATP
-in presence of this ATP, enzyme luciferase converts luciferin to oxyluciferin
-this conversion generates visible light which can be detected by a camera
-the amount of light generated is proportional to amount of ATP available and therefore indicates how many of same type if activated nucleotide were incorporated adjacent into complementary DNA strand
-unincorporated activated nucleotides are degraded by apyrase and the reaction starts again with another nucleotide
Bioinformatics
-a branch of biology called bioinformatics has grown out of this research to store huge amounts of data generated
-it would have been impossible to store and analyse these data prior to computers and microchips
-software packages are specially designed for this purpose
What was discovered as a result of human genome project
-genome was sequenced by 2003
-scientists learnt human genome contained only about 24,000 genes
Genome comparisons between individuals and species
-whole genome sequencing determines complete DNA sequence of an organisms genome- in case of eukaryotic cells that is the genetic material of chromosomes, mitochondria and chloroplasts
-sequenced genomes are stored in gene banks
Comparison between species
-when human genome was compared with those of other species it became clear that few human genes are unique to us
-most of our genes have counterparts in other organisms
-we share 99% of our genes with chimpanzees
-this verifies that genes work well tend to be conserved by evolution
-sometimes as evolution progresses some genes are coopted to perform new tasks
-tiny changes to gene in humans called FOXP2 allows speech
-many of the differences between organisms are not because the organisms have totally different genes but because some of their shared genes have been altered and now work in subtly different ways
-some changes to regulatory regions of DNA that do not code directly for proteins have also altered expression of genomes- regulatory and coding genes interact in such ways that without increasing number of genes, the number of proteins may be increased
Evolutionary relationships
-comparing genomes of organisms thought to be closely related species has helped confirm their evolutionary relationships or has led to new knowledge about the relationships and in some cases to certain organisms being reclassified
-DNA from bones and teeth of some extinct animals can be amplified and sequenced so the animals evolutionary history can be verified
Variation between individuals
-all humans are genetically similar
-except for rare cases where a gene has been lost by depletion of part of a chromosome we all have the same genes, but different alleles
-about 0.1% of our DNA is not shared with others
-the places on DNA where these substitutions occur are called single nucleotide polymorphisms or SNPs
-some have no effect on the protein, some can alter a protein or alter the way a piece of RNA regulates the expressions of another gene
-methylation of certain chemical groups in DNA plays a major role in regulating gene expression in eukaryotic cells
-methods to map to methylation of whole human genomes can help researchers understand development of certain diseases, for example cancer and why they may or may not develop in genetically similar individuals
-the study of this aspect of genetics is called epigenetics
Predicting amino acid sequences of proteins
-determining sequence of amino acids within protein is laborious and time consuming
-however if researchers have organisms genome sequenced and know which gene codes for a specific protein by using knowledge of which base triplets code for which amino acids, they can determine the primary structure of proteins
-researchers need to know which part of gene codes for exons and which codes for introns
Synthetic biology
-synthetic biology is an interdisciplinary science concerned with designing and building useful biological devices and systems
-it encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics
-its ultimate goal may to build engineered biological systems that store and process information, provide food, maintain human health and enhance environment
-sequences of DNA found by analysing genomes provide potential building blocks for synthetic biologist to build devices
Examples of synthetic biology
-information storage= scientists can encode a vast amount digital information onto single strand of synthetic DNA
-production medicines= E.coli and yeast have both been genetically engineered to produce precursor of good antimalarial drug, only available by extracting it from certain parts of Artemisia plants at particular times to its life cycle
-novel protein= designed proteins produced for example one similar to haemoglobin and binds to oxygen but not to carbon monoxide
-biosensors= modified bioluminescent bacteria, placed on a coating of microchip, grow if air polluted with petroleum pollutants
-nanotechnology= material can be produced for nanotechnology e.g. amyloid fibres for making biofilms - for functions such as adhesion
Bioethics
-synthetic biology raises issues of ethics and biosecurity
-extensive regulations are already in place, due to 30-40 years of using genetically modified organisms
-there are many advisory panels and many scientific papers have been written on how to manage risks
-synthetic biology is not about making synthetic life forms from scratch but about potential for new systems with rewards and associated risks to be managed
Development of DNA profiling
-Alec Jeffreys was locating tandem repeat sequences of DNA
-tandem repeats are repetitive sequences of DNA that do not code for proteins
-they may be between 10 and 100 base pairs long and they all feature the same core sequences GGCAGGAXG where X can be any of the four nucleotide bases
-tandem repeats occur at more than 1000 locations in genome and in each of these places they may be repeated a random number of times
-some types are highly variable and called variable number tandem repeats (VNTRs)
DNA profiling procedure
1) Data is obtained from the individual- either by mouth swab, from saliva on tooth brush, blood or hair or in case of ancient remains, bone
2) DNA is then digested with restriction enzymes. These enzymes cut the DNA at specific recognition sites. They will cut into fragments which will vary in size to each individual
3) the fragments are separated by gel electrophoresis and stained. larger fragments travel shortest distance in gel
4) a banding pattern can be seen
5) DNA to which individuals is being compared is treated with same restriction enzymes and subjected to electrophoresis
6) the banding patterns of DNA samples can then be compared
Types of DNA analysed
-the first method involved in restriction fragment length polymorphism analysis- method is no longer used as is laborious
-today short tandem repeat (STR) sequences of DNA are used
-these are highly variable short repeating length of DNA
-the exact number of STR varies from person to person
-each STR is polymorphic but the number of alleles in the gene pool is small for each one
-technique is very sensitive and even trace of DNA left when someone touches object can produce a result
-samples must be treated carefully to avoid contamination
-DNA can be stored for many years if criminal case unsolved- can later be used to assess new evidence
APPLICATIONS OF DNA PROFILING- forensic science
-DNA profiling has brought about convictions, establish innocence of many people and of people previously wrongly convicted
-e.g. identify nazi war criminals hiding in south america
-identify victims body parts after air crashes, terrorist attacks etc
-identify individuals remains
APPLICATIONS OF DNA PROFILING- maternity and paternity tests
-half of every child genetic information comes from mother and half from father
-hence short tandem repeat fragments come from mother and father
-comparing DNA profiles of mother, father and child can therefore establish maternity and/or paternity
APPLICATIONS OF DNA PROFILING - analysis of disease
-protein electrophoresis can detect type of haemoglobin present and aid diagnosis of sickle cell anaemia
-a varying number of repeat sequences for condition such as Huntington disease can be detected by electrophoresis
Principles of the polymerase chain reaction (PCR)
-Kay Mullis developed the technique the polymerase chain reaction to amplify (increase amount) of DNA, enabling analysis
-PCR soon became incorporated into forensic DNA analysis and into protocols for analysis of DNA for genetic diseases
-the PCR is artificial replication of DNA- it relies upon:
-DNA is made of two antiparallel backbone strands
-each DNA strand has a 5’ end and 3’ end
-DNA grows only from the 3’ end
-base pairs pair up according to complementary base pairing rules A with T, G with C
How does PCR differ from DNA replication
-only short sequences of up to 10,000 base pairs of DNA can be replicated not entire chromosomes
-it requires addition of prime molecules to make process start
-cycle of heating and cooling is needed to separate DNA strands, bind primers to strands and for DNA strands to be replicated
PCR process
1) sample DNA mixed with DNA nucleotides, primers, magnesium ions and enzyme Taq polymerase
-Taq polymerase is obtained from thermophilic bacteria and therefore stable at high temperatures
2) mixture heated to around 94-96C to break hydrogen bonds between complementary nucleotide base pairs and this denature double stranded DNA into 2 single strands of DNA
3) mixture cooled to 68C so primers can anneal (bind to hydrogen bonding) to one end of each single stranded DNA
-this gives small section of double stranded DNA at end of each single stranded molecule
4) Taq polymerase enzymes can now bind to end where there is double stranded DNA
-72C optimum for this enzyme
5)temperature raised to 72C keeping DNA as single strands
6) Taq DNA polymerase catalyses addition of DNA nucleotides to single stranded DNA molecules starting at end with primer and proceeding in 5’ to 3’ direction
7) when Taq polymerase reaches other end of DNA molecules a new double strand of DNA has been generated
8) whole process begins again and repeats for many cycles
Applications of the PCR
-tissue typing: donor and recipient tissues can be typed prior to transplantation to reduce risk of rejection of transplant
-detection of oncogenes: if type mutation involved in specific patient cancer found, medication better tailored to that patient
-detecting mutations: sample DNA analysed for presence of mutation that leads to genetic disease. Parents can test to see if carry recessive allele for particular gene e.g. fetal cells may be obtained from mothers blood stream for prenatal genetic screening
-identifying viral infections: sensitive PCR tests can detect small quantity of viral genome amongst host cell DNA e.g. for HIV, hepatitis C
-monitoring spread infectious disease: spread of pathogens through wild or domestic populations or from animals to humans can be monitored and emergence of new virulent sub types can be identified
-forensic science: small quantities of DNA can be amplified for DNA profiling to identify criminals or ascertain parentage
-research: amplifying DNA from extinct ancient sources for analysis and sequences
ELECTROPHORESIS: separating DNA
-electrophoresis is used to separate different sized fragments of DNA
-it can separate fragments that differ only by one base pair and is widely used in gene technology to separate DNA fragments for analysis and identification
-the technique uses and agarose gel plate covered by buffer solution
-electrodes are placed in each end of tank so when connected to power supply and electric current can pass through the gel
-DNA has an overall negative charge due to its many phosphate groups and the fragments of DNA all have similar surface charge regardless of size
ELECTROPHORESIS: separating proteins
-principle for separating proteins is same as for separating fragments of DNA but often carried out in presence of charged detergent such as sodium dodecyl sulfate (SDS) which equalises surface charge on molecules and allows proteins to separate as they move through the gel according to their molecular mass
What can separating proteins be used for
-technique can be used to analyse types of haemoglobin proteins for diagnosis of conditions such as
-sickle cell anaemia where patient has haemoglobin S not normal A
-aplastic anaemia, thalassaemia and leukaemia where patients have higher than normal amounts of fetal haemoglobin (F) and lower than normal amounts of haemoglobin A
Using DNA probes
-a DNA probe is a short single stranded length of DNA that is complementary to section of DNA being investigated
-probe may be labelled using
-a radioactive marker usually with 32P in one of phosphate groups in probe strand
-once probe has annealed by complementary base pairing to piece of DNA it can be revealed by exposure to photographic film
-or a fluorescent marker that emits a colour on exposure to UV lighr
-fluorescent markers may also be used in automated DNA sequencing
What are DNA probes used for
-useful in locating specific DNA sequences e.g.
-to locate specific gene needed for use in genetic engineering
-to identify same gene in variety of different genomes from different species when conducting genome comparison studies
-to identify presence or absence of specific allele for particular genetic disease or that fives susceptibility to particular condition
Microarrays
-scientists can place a number of different probes on fixed surface known as DNA microarray
-applying DNA under investigation to surface can reveal presence of mutated alleles that match fixed probes because sample DNA will anneal to any complementary fixed probes
-sample DNA must first be broken into smaller fragments and may also be amplified using PCR
-a DNA microarray can be made with fixed probes, specific for certain sequences found in mutated alleles that cause genetic diseases
-references and test DNA samples are labelled with fluorescent markers
-where test subject and reference marker both bind to particular probe, the scan reveal fluorescence of both colours, indicating presence of particular sequence in test DNA
Principles of genetic engineering
-genetic engineering is also known as recombinant DNA technology because it involves combining DNA from different organisms
-also called genetic modification
-genes are isolated from one organism and inserted to another organism using suitable vectors
1) obtaining the required gene
2) a copy of gene is placed inside vector
3) vector carries gene into recipient cell
4) recipient expresses novel gene
1 required gene is obtained
-mRNA can be obtained from cells where the gene is being expressed
-an enzyme reverse transcriptase, can then catalyse the formation of single strand of complementary DNA (cDNA) using mRNA as template
-the addition of primers and DNA polymerase can make this cDNA into double stranded length of DNA whose base sequence codes for the original protein
-if scientists know the nucleotide sequence of gene then the gene can be synthesised using automated polynucleotide synthesiser
-if scientists know the sequence of the gene the can design PCR primers to amplify the gene from genomic DNA
-a DNA probe can be used to locate a gene within genome and gene can be cut out using restriction enzymes
- Placing gene into vector
-plasmids can be obtained from organisms such as bacteria and mixed with restriction enzymes that will cut the plasmid at specific recognition sites
-cut plasmid has exposed unpaired nucleotide bases called sticky ends
-if free nucleotide bases complementary to sticky ends of plasmid are added to ends of gene to be inserted then gene cut and plasmid should anneal
-DNA ligase catalyses annealing
-gene may be sealed into attenuated virus that could carry it into host cell
- Getting vector into recipient cell
-DNA does not easily cross recipient cells plasma membrane
-various methods can be used to aid the process:
-heat shock treatment- if bacteria subjected to alternating periods of cold 0C and heat 42C in presence of calcium chloride, their walls and membranes will become more porous and allow in the recombinant vector. This is because positive calcium ions surround negatively charged parts of both DNA molecules and phospholipids in cell membrane, this reducing repulsion between foreign DNA and host cell membranes
-electroporation- high voltage pulse applied to cell to disrupt membrane
-electrofusion- electrical fields help introduce DNA into cells
-transfection- DNA can be packaged into bacteriophage which can then transfect into host cell
-T1 (recombinant) plasmids are inserted into bacterium Agrobacterium tumefaciens which then infects some plants and naturally inserts its genome into host cell genomes
- Direct method introducing gene into recipient
-if plants not susceptible to A. tumefaciens then direct methods can be used
-small pieces of gold or tungsten coated with DNA and shot into plant cells
-this is called a gene gun
Reverse transcriptase
-retroviruses such as HIV which contain RNA that they inject into host genome have reverse transcriptase enzyme that catalyses the production cDNA using their RNA as template
-this is reverse of transcription
-these enzymes are useful for genetic engineering
Restriction enzymes
-bacteria and archaea have restriction enzymes called restriction endonucleases to protect them from attack by phage viruses
-these enzymes cut up foreign viral DNA by a process called restriction preventing the viruses from making copies of themselves
-the prokaryotic DNA is protected from action of endonucleases by being methylated at recognition sites
-the restriction endonucleases are named according to bacterium from which they have been obtained
-the first used was obtained from E.coli
-restriction enzymes are useful to molecular biology and biotechnology as molecular scissors as they recognise specific sequences within length of DNA and cleave molecular there
-some make a staggered cut leaving sticky ends
-other make a cut that leaves blunt ends
-the enzymes always recognise a palindromic sequence; reading two strands of DNA in same orientation, sequences of bases is same
-some restriction endonucleases need magnesium ions as cofactors
Ligase enzymes
-DNA ligase enzymes used in molecular biology to join DNA fragments
-it catalyses condensation reactions that join sugar groups and phosphate groups of DNA backbone
-these enzymes catalyse such reactions during DNA replication in cells and are also used in PCR
Insulin from GM bacteria
1)adding reverse transcriptase enzymes makes single strand of cDNA and treatment with DNA polymerase makes double strand- the gene
2) addition of free unpaired nucleotide at end of DNA produces sticky ends
3) now with help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bacteria
-these are now called recombinant plasmids as they contain inserted DNA
4)E.coli bacteria are mixed with recombinant plasmids and subjected to heat shock in presence of calcium chloride ions so they will take up plasmids
Safety of genetic engineering
-because transformed/ transgenic bacteria have resistance to some antibiotics we don’t want them to escape laboratories into wild
-they also have gene knocked out meaning they cannot synthesise particular nutrient
-they survive in lab where they are given that nutrient in growth medium but will not survive outside these conditions
ISSUES GENETIC MANIPULATION: microorganisms
+GM microorganisms can make human insulin to treat all diabetics (not possible with pig insulin)
+make human growth hormone to treat children with pituitary dwarfism
-microorganism could escape into wild and transfer marker genes for antibiotic resistance to other bacteria - HOWEVER, cannot synthesise essential nutrient so will not survive outside lab
ISSUES GENETIC MANIPULATION: plants
+1985 tobacco plants were genetically modified to produce toxin normally produced by a bacterium Bacillus thurigiensis
+this toxin Bt has been used by organic farmers as pesticide as it is toxic to insects
+because bacterial gene Bt inserted into some crop plants, the GM plants produced the toxin eliminating need to spray it around the environment possibly contaminating other organisms
-Bt is toxic to monarch butterflies
ISSUES GENETIC MANIPULATION: soya beans
+GM soya beans, resistant to herbicide, were produced so weeds competing with soya plants could be killed with herbicide
-possible risks include potential for gene for herbicide resistance to pass into weeds producing superweeds
ISSUES GENETIC MANIPULATION: golden rice
+golden rice was genetically modified to contain a gene from daffodils so that beta carotene would be present in rice grains to prevent loss of eyesight due to vitamin A deficiency
-some people concerned farmers would have to buy seed every year
ISSUES GENETIC MANIPULATION: plantains/ bananas
+local biotechnology company in Kenya is producing plantains that are nutritionally enhanced to contain more zinc in areas where people eat very little meat; they may be deficient in zinc an important enzyme cofactor and essential for regulating insulin secretion
-some people fear eating food that contains foreign DNA and worry that inserted genes will somehow be expressed in us
ISSUES GENETIC MANIPULATION: crops resistant to pests
+biotechnology company producing crop resistant to pests so when farmers sow seeds they do not need to use pesticides- this is better for environment and less exposure to farmers
-concerns local farmers might not want GM seeds and not have choice to buy non GM seeds
ISSUES GENETIC MANIPULATION: pathogens
+ viruses genetically modified to have no virulence can be used to make vaccines as they still have antigens on their surface- this reduces chance of vaccine making recipient ill
+modified viruses can also be used as vectors in gene therapy
-allele may be used inserted into genome in way that increases risk of cancer or interfere with gene regulation
ISSUES GENETIC MANIPULATION: mice
+GM mice bred for medical research and used to develop therapies for therapies for breast and prostate cancer
-some people object to use of animals for medical testing
ISSUES GENETIC MANIPULATION: pharmaceutical proteins
+genes for human pharmaceutical proteins such as alpha antitrypsin to treat hereditary emphysema can be inserted into goats or sheep and human protein they express into milk is harvested
-concerns for welfare of GM sheep and goat
ISSUES GENETIC MANIPULATION: silk
+genes for spider silk inserted into goat- produce spider silk protein in their milk for use in cables, stitches, bullet proof vests etc
-concerns for welfare of GM goats
Principle of gene therapy
-basic principle of gene therapy is to insert functional allele of particular gene into cells that contain only mutated and non functioning alleles of that gene
-if inserted allele is expressed then individual will produce functioning protein and no longer have symptoms associated with genetic disorder
-knowledge gained from Human Genome project has led to further possibilities such as using interference RNA to silence genes by blocking translation
-interference RNA has been used to treat cytomegalovirus in AIDs patients by blocking replication of said virus
Somatic cell gene therapy
-some metabolic disorders such as cystic fibrosis occur when individual inherits two faulty recessive alleles for particular gene
-as result, differentiated cells where this gene should normally be expressed lack the protein product of that gene
-if functioning alleles for this gene can be put into specific cells so that these cells can make the protein, these cells will function normally
-various methods are available for delivering functioning alleles to the patients body cell
-somatic cell gene therapy affects only certain cell types
-alterations made to patients genome in those cells are not passed to patients offspring
Liposomes
-patients with cystic fibrosis lack functioning CFTR gene
-alleles which are lengths of DNA can be packaged within small spheres of lipid bilayer to make liposomes
-if these are placed into aerosol inhaler and sprayed into noses of patients, some will pass through plasma membrane of cells lining respiratory tract
-if they also pass through nuclear envelope and insert into host genome, host cell will expresses CFTR protein- a transmembrane chloride ion channel
-epithelial cells lining respiratory tract are replaced every 10-14 days so this treatment has to repeated at regular short intervals
Viruses
-viruses have been used as vectors
-if virus that usually infects humans is genetically modified so that is encases functioning allele to be inserted into patient whilst at same time being made unable to cause disease, it can enter recipient cells taking allele with it
-there are potential problems with using viruses as gene delivery agents:
-viruses still may provoke immune or inflammatory response
-patient may become immune to virus, making subsequent deliveries difficult
-virus may insert allele into patient genome in location that disrupts gene involved in regulating cell division, increasing cancer risk
-virus may insert allele into patients genome in location that disrupts regulation of expression of other genes
Artificial chromosomes
-research done for possibility of inserting genes into artificial chromosome that would co-exist with other 46 chromosomes in target cell
Germ line gene therapy
-germ line therapy involves altering genome of gametes or zygotes
-not only will all cells of that individual be altered, their offspring may also inherit the foreign allele
-this type of therapy has potential to change genetic makeup of many people, descendants of original patient, non of whom would have given consent
-also concerns about how genes may inserted- may find their way into a location that could disrupt expression or regulation of other genes or increase risk of cancer
-strict guidelines drawn up by regulatory bodies and ethic committees consider germline therapy for humans to be ethically impossible
