GENOME PROJECTS AND GENE TECHNOLOGIES

GENOME PROJECTICS AND MAKING DNA FRAGMENTS

GENOME - entire set of DNA including all genes in an organism. The Human Genome Project completed in 2003, mapped the entire sequence of the genome for the first time

Improvements in tech have allowed us to sequence the genome of a variety of organisms. These methods only walk on fragments of DNA.Sequencing the genome of simple organisms helps identify their proteins.

PROTENOME - all the proteins made by an organism.

Simple organisms (bacteria) dont have much non coding DNA so its relativelty easy to determine their protenome from the DNA sequence of their genome - can be useful in medical research and development e.g. identifying the protein antigens on the surface  of disease casuing bacteria and viruses can help in the development of  vaccines to prevent the disease.

More complex organisms contain large sections on non coding DNA & complex regulatory genes which determine when genes that code for particular proteins should be switched on or off. Its more difficult to translate their protenome - hard to find bits that code for proteins among non coding and regulatory DNA

In the past, sequencing methods were labour intensive, expensive and small scale. Now theyre automated, more cost effective and can be done on a large scale. With newer & faster tehcniques scientists can sequence whole genomes much more quickly. e.g. pryosequencing can sequence 400 million bases in 10 hours.

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GENOME PROJECTICS AND MAKING DNA FRAGMENTS

Recombinant DNA technology involves transferring fragments of DNA from one organism to another - because genetic code is universal & transcription and translation mechanisms are pretty similar, transferred DNA can be used to produce a protein in the cells of the recipient organisms (known as transferred organisms when they have transferred DNA) 

DNA fragments can be made in different ways 

USING REVERSE TRANSCRIPTASE;

  • most cells contain only 2 copies of each gene, but many mRNA copies (complimentary to the gene) 
  • mRNA molecules are used as templates to make lots of DNA. Reverse transcriptase makes DNA from RNA templates. DNA produced is called complimentary DNA. 
  • e.g. pancreatic cells produce insulin, they have many mRNA mol. complimentary to the insulin gene so reverse transcriptase can be used to make cDNA from the insulin mRNA. 
  • To do this, mRNA is first isolated from cells , then mixed with free DNA nucleotides and reverse transcriptase. RT uses the mRNA as a template to synthesis a new strand of cDNA.
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GENOME PROJECTICS AND MAKING DNA FRAGMENTS

USING RESTRICTION ENDONUCLEASE ENZYMES

  • some DNA sections have palindromic sequence of nucleotides - they consist of antiparallel base pairs .
  • restirction endonucleases are enzymes that recognise specific palindromic sequences (recognition sequences) and cut (digest) the DNA at these places.
  • different RE cut at different specific recognition sequences. Shpe of recogntiion sequence is complimentary to the enzymes active site. 
  • if RS are present at either side of the DNA fragment, you can use RE to seperate it from the rest of the DNA. 
  • DNA is incubated with the specific RE, which cuts the fragment out via a hydrolysis reaction.
  • sometimes this cut can leave 'sticky ends' - small tails of unpaired bases at each end of the fragment. These can be used to bind the DNA fragment to another that has complimentary sticky ends. 
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GENOME PROJECTICS AND MAKING DNA FRAGMENTS

USING A GENE MACHINE 

  • fragments of DNA can be synthesised from scratch without a pre exsisting template. A database contains the necessary info to produce the DNA fragment. This means the DNA doesnt have to exsist naturally - any sequence can be made.
  • the sequence required is designed (if not already exsisting)
  • first nucleotide in the sequence is fixed to some sor of support e.g. a bead
  • nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups - these make sure that the nucleotides are joined at the right potins to prevent unwanted branching.
  • short sections of DNA (oligonucleotides) roughly 20 long are produced. Once complete they are broken off from the support and all protecting groups are removed - these can then be joined together to make longer DNA fragments. 
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AMPLIFYING DNA FRAGMENTS

In Vivo amplification involves transforming host cells. Once the DNA fragment is isolated it needs to be amplified (make loads of copies) so theres a sufficent quantity to work with. One method of doing this in to use in vivo cloning - copies of the DNA fragment are made inside a living organism.

STEP 1 - DNA FRAGMENT IS INSERTED INTO A VECTOR

  • DNA fragment is inserted into vector DNA - a vector is something thats used to transfer DNA into a cell, they can be plasmids or bacteriophages (viruses that infect bacteria)
  • Vector DNA is cut open using same RE that was used to isolate the DNA so the sticky ends of the vector are complimentary to the sticky ends of the DNA fragment containing the gene. 
  • Vector DNA and DNA fragments are mixed together with DNA ligase - this joins sticky ends of the DNA fragment to the sticky ends of the vector DNA. This is called ligation.
  • The new combination of the bases in the DNA (vector DNA & DNA fragment) is known as recombinant DNA.
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AMPLIFYING DNA FRAGMENTS

STEP 2 - VECTOR TRANSFERS THE DNA FRAGMENT INTO HOST CELLS

  • vector with the recombinant DNA is used to transfer the gene into cells (host cells)
  • if a plasmid vector is used, host cells have to be persuaded to take in plasmid vector and its DNA. e.g. host bacterial cells are placed in ice cold calcium chloride to make their cell walls more permable. Plasmids are added and mix is heat shocked which encourages cells to take in the plasmids.
  • with a bacteriophage vector, bacteriophage will infect the host bacterium by injecting its DNA into it. Phage DNA then integrates into the bacterial DNA.
  • host cells that take up the vectors containing the gene are known to be transformed. 

To produce proteins you need promoter and terminator regions (the vector must contain these). Promotor regions are DNA seqeunces that tell the enzymes RNA polymerase when to start prodcuing mRNA. Terminator regions tell it when to stop. Without the right promoter region, DNA fragment wont be transcribed by the host cell and a protein wont be made. Promotor and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment 

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AMPLIFYING DNA FRAGMENTS

STEP 3 - IDENTIFYING TRANSFORMED HOST CELLS

  • around 5% of host cells will take up the vector and its DNA.
  • Marker genes can be inserted into vectors as the same time as the gene to be cloned. Any transformed host clels will contain the gene to be cloned and the marker gene.
  • host cells are grown on agar plates, each cell divides and replicates its DNA, creating a colony of cloned cells. Transfromed cells will produce colonies where all cells contain the cloned gene and the marker gene.
  • marker gene can code for antibiotic resistance - host cells are grown on agar plates containing a specific antibiotic, only transformed cells that have the marker gene will survive and grow. Or it can code for flourescence - when the agar plate is placed under a UV light only transformed cells will show.
  • Identified transformed cells are allowed to grow more, producing many copies of the cloned gene.
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AMPLIFYING DNA FRAGMENTS

IN VITRO amplification uses the polymerase chain reaction (PCR). Dna fragments can also be amplified using in vitro cloning - copies of the DNA fragments are made outside of a living organism using the PCR.

  • a reaction mixture is set up that contains the DNA sample, free nucleotides, primers (short pieces of DNA that are complimentary to the bases at the start of the fragment you want)and polymerase.
  • the mix is heated to 95 degrees to break the H bonds between the two strands of DNA.
  • mix is then cooled to between 50-65 degrees so that primers start to bind (anneal) to the strands.
  • mix is then heated again to 72 degrees so DNA polymerase can work
  • DNA polymerase lines up free DNA nucleotides alongside each template strand. Specific base pairing means new complimentary strands are formed.
  • 2 new copies of the fragment of DNA are formed and one cycle of PCR is complete.
  • cycle starts again with the mix being heated. All four strands are used as templates,
  • each PCR cycle doubles the amount of DNA. 
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USING RECOMBINANT DNA TECH

Transformed organisms are made using recombinant DNA tech. Microorganisms, plants and animals can all be transformed using recombinant DNA tech - genetic engineering. Transformed microorganisms can be made using the same tech as in vivo cloning. e.g. foreign DNA can be inserted into microorganisms to produce lots of a useful protein e.g. insulin;

  • DNA fragment containing the insulin gene is isolated using a technique 
  • the fragment is inserted into a plasmid vector.
  • the plasmid containing the recombinant DNA is trasnferred into a bacterium
  • transformed bacteria are identified and grown
  • the insulin produced from the cloned gene is extracted and purified. 

Transformed plants can also be produced. A gene that codes for a desireable protein is inserted into a plasmid. Plasmid is added to a bacterium and the bacterium is used as a vector to get the gene into the plant cells. If the right promoter region has been added along with the gene the transformed cells will be able to produced the desired protein. 

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USING RECOMBINANT DNA TECH

Transformed animals can be produced - a gene that codes for a desireable protien is inserted into an early animal embryo/egg cells of a female. If inserted into an early embryo all the body cells will contain the gene. If from eggs, all the cells of her offspring will contain the gene. 

Promoter regions that are only activated in specific cell types can be used to control exactly which of an animals body cels the protein is produced in. If its only produced in certain cells it can be harvested easier. Producing the protein in the wrong cells could damage the organism. 

Transformed organisms can be used in a variety of ways;

  • AGRICULTURE - crops can be transformed so they give higher yeilds/more nutritious which can reduce risk of famine and malnutrition. Can also be transformed to have pest resistance which reduces cost and any environmental problems with using pesticides. e.g. golden rice has been tranformed with one gene from a maize plant and one from a soil bacterium which produce beta-carotene which produces vitamin A - can help combat vitamin A deficency. 
  • INDUSTRY - industrial processes often use bio catalysts (enzymes)| - these can be produced from transformed organisms, so they produce large quantities for less money. e.g. chymosin is used in cheese making it used to be made from remet (found in cow stomach) but can now be produced from transformed organisms = large quantities, cheaply and dont have to kill cows.
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USING RECOMBINANT DNA TECH

  • MEDICINE- drugs and vaccines are produced by transformed organisms. They can be made quickly,cheaply and in large quantities. e.g. insulin is used to treat type 1 diabetes and used to come from animals - because its not human it doesnt work as well. Human insulin is now made from transformed microorganisms using a cloned insulin gene, 

There are ethical,financial and social issues with the use of recombinant DNA tech;

  • agriculture - some are concerned about possibility of superweeds - resistant to herbicides. These occur if transformed crops interbreed with wild plants. There could be an uncontrolled spread of recombinant DNA with unknown consequences.
  • industry -some consumer markets wont import GM foods and products. This can cause a loss to producers.
  • medicine - some worry this tech will be used unethically e.g. designer babies. 

Recombinant DNA also creates ownership issues;

  • there is debate about who owns genetic material once it has been removed from the body. Some say the individual holds the right to their own genetic info, other argue that value is created by the researcher who uses it to develop a medicine or in diagnosis. 
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USING RECOMBINANT DNA TECH

Recombinant DNA tech has many potential humanitarian benefits;

  • agricultural crops could be produced that help reduce the risk of famine and malnutrition
  • transformed crops could be used to produce useful pharmaceutical products (vaccines) making drugs available to more people
  • medicine can be produced more cheaple, so more can afford them
  • recombinant DNA tech has the potential to be used in gene therapy to treat human diseases.

Gene therapy;

  • involves altering the defective genes inside cells to treat genetic disorders and cancer
  • how this is done depends on whether the disorder is caused by a mutated dominant allele or two mutated recessive alleles.
  • if its two recessive alleles you can add a dominant working allele to make up for them (you supplement the faulty ones)
  • if its a dominant allele you can silence the dominant allele (stick DNA in the middle so it doesnt work any more
  • allele is inserted into cells using vectors. Different vectors can be used e.g. altered viruses,plasmids or liposomes 
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USING RECOMBINANT DNA TECH

Two types of gene therapy;

Somatic therapy - involves altering the alleles in body cells, mostly those affected by the disorder. e.g. CF damages the respiratory system so somatic therapy for CF targets the epithelial cells lining the lungs. Somatic therapy doesnt affect the individuals sex cell, offspring could still inherit the disease.

Germ line therapy - involves altering the alleles in the sex cells. Every offspring cell will be affected by the gene therapy and they wont suffer from the disease. This is currently illegal.

Many ethical issues 

  • some are worried that the tech could be used in ways other than for medical treatment. e.g. cosmetic effects of aging.
  • more harm than good could be done by using the tech e.g. risk of overexpression of genes - genes produces too much of the missing protein.
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GENE PROBES AND MEDICAL DIAGNOSIS

DNA probes can be used to locate specific alleles of genes (e.g. on chromosomes) or to see if a persons DNA contains a mutated allele that causes a genetic disorder. DNA probes are short strands of DNA, they have specific base sequence thats complimentary to the base sequence of a part of a target allele. DNA probe will bind to the target allele if its present in a sample of DNA. A DNA probe has a label attached, so it can be detected, can be either radioactive (detected using X-RAY film) or flourescent (detected using UV light). Heres how its done;

  • a sample of DNA is digested into fragments using restriction enzymes and seperated using electrophoresis
  • the seperated DNA fragmentss are then transferred to a nylon membrane and incubated with the flourescently labelled DNA probe
  • if the allele is present the DNA probe will hybridise to it. 
  • the membrane is then exposed to UV light and if the gene is present there will be a flourescent band.
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GENE PROBES AND MEDICAL DIAGNOSIS

alternatively the probe can be used as part of a DNA microarray;

  • a DNA microarray is a glass slide wiith microscopic spots of different DNA probes attached in rows
  • a sample of flourescently labelled human DNA is washed over the array
  • if the human DNA contains any DNA sequences that match any of the probes, it will stick to the array
  • the array is then washed to remove any labelled DNA that hasnt stuck to it
  • the array is then visualised under UV light - any labelled DNA attached to a probe will show up - this means the persons DNA contains that specific allele.

To produce a DNA proobe, you first need to seqeucene the allele that you want to screen for. You then use PCR to produce multiple complimentary copies of part of the allele (probes)

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GENE PROBES AND MEDICAL DIAGNOSIS

The results of screening can be used for genetic counselling. Genetic counselling is advising patients and their relatives about the risks of genetic disorders. It involves advising people about screening and explaining. Screening can help to identify is someones the carrier of a mutated allele, the type of mutated allele and the most effective treatment. 

The results of screening can also be used for personalised medicine. As your genes determine how your body responds to cretain drugs. Different people respond to different drugs in different ways. Personalised medicines are tailored to an individuals DNA. If doctors have your genetic information they can use it to predict how you will respond to different drugs and only prescribe the ones that will be the most effective for you. 

Screening using DNA probes has lots of uses. For example, it can be used to help identify inherited conditions e.g. huntingtons dieases affects the nervous system and symptoms dont present until 30-50 years. People with a family history of the disease may choose to be screened for the mutated allele to see if they have inherited it. It can also be used to help identify health risks. e.g. inheriting particular mutated alleles increases your risk of developing certain cancers. If a person knows they have these alleles it might help them to make choices that could reduce the risk of developing cancers. However some are concerned that genetic screening may lead to discrimination by insurance companies.

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GENETIC FINGERPRINTING

Not all of an organisms genome codes for proteins, some consists of variable number tandem repeats - base sequences that dont code for proteins and repeat next to each other over and over. The number of times this repeat happens varies from person to person, so the length of these seqeunces in nucleotides differs too. The repeated seqeunces occur in lots of places in the genome. The number of times a sequence is repeated at different places in their genome can be compared between individuals - genetic fingerprinting. The probabilitiy of 2 individuals having the same genetic fingerprint is v low because the chance of 2 having the same no. of VNTR's at each place theyre found in DNA is very low. 

Genetic fingerprinting is used to determine relationships (we inherit VNTRS base seqeunces from our parents, the more bands on a genetic fingerprint that match the more closely related two people are) and variability within a population (the greater number of bands that dont match on a genetic fingerprint the more genetically different people are. You can compare the number of repeats at several places in the genome for a population to find out how genetically varied that population is) 

Genetic fingerprinting can be used in animal and plant breeding, they can prevent interbreeding which decreases the gene pool. Interbreeding can also lead to an increased risk of genetic disorders leading to health, productivity and reproductive problems. Genetic fingerprints can be used to identify how closely-related 2 individuals are, the more similar their genetic fingerprint will be. The least related will be bred together. 

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GENETIC FINGERPRINTING

Electrophoresis seperates DNA fragments to make a genetic fingerprint so they can be compared between individuals;

  • a sample of DNA is obtained (from blood, saliva)
  • PCR is used to make many copies of the areas of DNA that contain the VNTRS - primers are used that bind to either side of these repeats so the whole repeat is amplified
  • you end up with DNA fragments where the length (in nucleotides) corresponds to the number of repeats the person has at each specific position. 
  • a flourescent tag is added to all DNA fragments so they can be viewed under UV light. The DNA fragments then undergo electrophoresis
  • DNA mixed is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity. A current is then passed through the gel - DNA fragments are -ve so move towards +ve electrode at the far end of the gel. Small fragments move faster and travel further through the gel, the fragments seperate according to size. 
  • DNA fragments are viewed as bands under UV light - this is the genetic fingerprint
  • two genetic fingerprints can be compared -  if both have a band at the same location on the gel it means they have the same no. of nucleotides & same number of VNTRs at that place - its a match.
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GENETIC FINGERPRINTING

Genetic fingerprinting can be used in forensic science to compare samples of DNA collected from crime scenes (e.g. DNA from blood,semen, skin cells) to samples of DNA from possible suspects which could link them to crime scenes;

  • DNA is isolated from all the collected samples
  • each sample is replicated using PCR
  • PCR products are run on an electrophoresis gel and the genetic fingerprints produced are compared to see if any match
  • if samples match, it links a person to the crime scene.

Genetic fingerprinting can also be used in medical diagnosis. In medical diagnosis a genetic fingerprint can refer to a unique pattern of several alleles. It can be used to diagnose genetic disorders and cancer - its useful when the specific mutation isnt known/where several mutations could have caused the disorder as it identifies a broader, altered genetic pattern. 

e.g. Genetic fingerprinting can be used to diagnose sarcomas (types of tumours) Conventional methods of identifying a tumour only show the physcial differences between tumours. Now the genetic fingerprint of a known sarcoma (different mutated alleles) can be comapred to the genetic fingerprint of a patients tumour. If theres a match, the sarcoma can be specifically diagnosed and treatment can be targeted. 

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GENETIC FINGERPRINTING

Genetic fingerprinting can be used in forensic science to compare samples of DNA collected from crime scenes (e.g. DNA from blood,semen, skin cells) to samples of DNA from possible suspects which could link them to crime scenes;

  • DNA is isolated from all the collected samples
  • each sample is replicated using PCR
  • PCR products are run on an electrophoresis gel and the genetic fingerprints produced are compared to see if any match
  • if samples match, it links a person to the crime scene.

Genetic fingerprinting can also be used in medical diagnosis. In medical diagnosis a genetic fingerprint can refer to a unique pattern of several alleles. It can be used to diagnose genetic disorders and cancer - its useful when the specific mutation isnt known/where several mutations could have caused the disorder as it identifies a broader, altered genetic pattern. 

e.g. Genetic fingerprinting can be used to diagnose sarcomas (types of tumours) Conventional methods of identifying a tumour only show the physcial differences between tumours. Now the genetic fingerprint of a known sarcoma (different mutated alleles) can be comapred to the genetic fingerprint of a patients tumour. If theres a match, the sarcoma can be specifically diagnosed and treatment can be targeted. 

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GENETIC FINGERPRINTING

Genetic fingerprinting can be used in forensic science to compare samples of DNA collected from crime scenes (e.g. DNA from blood,semen, skin cells) to samples of DNA from possible suspects which could link them to crime scenes;

  • DNA is isolated from all the collected samples
  • each sample is replicated using PCR
  • PCR products are run on an electrophoresis gel and the genetic fingerprints produced are compared to see if any match
  • if samples match, it links a person to the crime scene.

Genetic fingerprinting can also be used in medical diagnosis. In medical diagnosis a genetic fingerprint can refer to a unique pattern of several alleles. It can be used to diagnose genetic disorders and cancer - its useful when the specific mutation isnt known/where several mutations could have caused the disorder as it identifies a broader, altered genetic pattern. 

e.g. Genetic fingerprinting can be used to diagnose sarcomas (types of tumours) Conventional methods of identifying a tumour only show the physcial differences between tumours. Now the genetic fingerprint of a known sarcoma (different mutated alleles) can be comapred to the genetic fingerprint of a patients tumour. If theres a match, the sarcoma can be specifically diagnosed and treatment can be targeted. 

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