hi everyone and welcome to the final topic for ciee topic 19 genetic technology one of the hardest topics and in this video I'm going to go through everything you need to know much like my alevel notes do as well which go through all of the theory for the entire a level key terms key marking points and key examiner points as well so check that out in the link below if you want that boost to your a Lev grade but for now let's get into the content topic 19 the final topic is all about Gene technology so genetic technology and we're starting with the principles of genetic Technologies Rec combinant DNA Technologies is the technology of combining different organisms DNA together to create what is known as recombinant DNA and this is created by artificially joining together T DNA of two different species this is needed for scientists to manipulate and alter genes to improve industrial processes for medical treatment as well and then transferring DNA fragments between or within species is possible because the genetic code is universal transcription and translation also work the same in all organisms so what we mean by that is the same triplet of bases code for the same amino acid in all organisms so you can take the gene from one species in insert it into the DNA of another one and that same protein will still be created because the genetic codes universe Al and protein synthesis transcription and translation will work the same way genetic engineering is the manipulation of an organism's genetic material introducing a new Gene and therefore causing that organism to code for a protein that results in the desired traits or characteristics the process involves precise techniques to modify specific genes within the organism's genome and one common method in genetic engineering invol olves the transfer of a gene from one organism to another resulting in the expression of that Gene in the recipient organism this transfer genetic material can lead to the production of new proteins or the modification of existing cellular functions But ultimately it influences the phenotype of the organism so this is just a summary here of some Rec combinant DNA Technologies creating DNA fragments genetic FMA Printing and genetic counseling and we're going to be looking at this Branch first of all how you can create DNA fragments to do genetic engineering because all of this bit here in the blue is genetic engineering so DNA fragments which is so the first step in this recombinant DNA technology is to isolate the fragment of DNA that you want to insert into the DNA of another organism and there are three methods that can be used to isolate these fragments of DNA reverse transcription restriction and the nucleases and the gene machine reverse transcription this is when an enzyme makes DNA copies from mRNA and this enzyme naturally occurs in viruses such as HIV and it is reverse transcriptase so what you need to do is locate a cell that naturally produces the protein of Interest that would be because that cell will have lots of mRNA for that protein you can then remove that mRNA and combine it with the enzyme reverse transcriptase which is the enzyme which is going to join the DNA nucleotide bases to the MRNA sequence so we can see here we've got the MRNA molecule you would have complementary DNA nucleotides aligning opposite those mRNA bases and then you'd have a single strand of DNA created now you'd need to have a double strand so to make this DNA fragment double Stranded the enzyme DNA polymerase is then used and you'd actually then go on to use this in PCR to get multiple copies but the key things that you need to know is you start with an mRNA sequence you then add in DNA nucleotides and the enzyme reverse transcriptase and that will then create a comp complimentary single strand of DNA to that mRNA sequence then you add DNA polymerase to create a double strand now the advantage of this way to create your DNA fragment is the compliment DNA that is made is intron free because it was created from mRNA and mRNA has already had the introns spliced out restriction en and nucleases is another method and these are enzymes that cuts up DNA and these naturally occur within bacteria as a defense mechanism there are many different restriction enzymes that have an active site complementary in shape to a range of different DNA based sequences because that is what the active site is it's the part that is going to be complimentary in shape to the substrate which is a particular sequence of DNA bases and that section of DNA bases is described as the recognition sequence and in this way restriction enzymes or endonucleases cut DNA at specific locations some enzymes cut at the same location in the double strand and create a blunt end which isn't particularly useful because if you have a blun end it's really hard to join the DNA back together but other restriction endon nucleases cut to create staggered ends and exposed faes and that's what we can see here with this restriction end nucleas H three it cuts and creates this staggered end and therefore we have what's known as sticky ends called sticky because you've got these exposed bases which would then be able to align opposite their complimentary bases and in that way it's bit like they're sticky they can join together more easily so you get these cuts at palindromic sequences meaning it's the same one way as it is backwards the DNA base sequence and that's how we get our sticky ends which can then help to insert that DNA fragment into the DNA of another organism the G machine this is when DNA fragments are created in a lab using a computerized machine scientists first have to examine the protein of Interest they want this new organism to produce and in that way they identify the amino acid sequence of the protein and from that they then work backwards and work out the MRNA and DNA SE quence that would be required to code for that amino acid sequence that DNA sequence is then entered into a computer and checked for Bio safety and biocurity that the DNA being created is safe and ethical to produce the computer then create small sections of overlapping single strands of nucleotides that make up the gene called oligonucleotides oligonucleotides can then be joined to create the DNA for an entire Gene once you've done that PCR is then used to amplify the quantity and to make it double stranded so this process using a GM machine is very quick it's accurate and you can also design it so it's intron free so that's how we create our DNA fragments next time we're going to have a look at how you can clone those fragments so you get lots of copies of it so you could do this in Vivo which means inside of a living thing so we've talked about how you create your DNA fragment of Interest now we can look at how you insert that DNA fragment into a vector which is an organism that will carry the DNA into the host and the host is the organism whose DNA you are modifying so once we've actually used those restriction endonucleases to create our DNA fragments and we've got our sticky ends we actually have to modify the DNA so it can be used in transcription so we have to add a promoter region which is added at the start of the DNA fragment and this is a sequence of DNA which is The Binding site for RNA polymerase which will therefore enable transcription to occur and we have to also add a Terminator region which is added at the end of the Gene and this causes RNA polymerase to detach and stop transcription so only one gene at a time is copied into mRNA once we've done that then we insert this DNA into a vector and a vector is something that carries this DNA fragment into the host cell plasmids are the most common vectors which are used and plasmids are Loops of DNA that are found in some bacteria so that circular DNA which is separate from the main bacterial genome only contains a few genes so the way that we insert it then is the plasmid is cut open using the same restriction end nuclease that was used to cut out the gene of interest that creates the same sticky ends and therefore you're going to have sticky ends complementary to each other on the DNA fragment and on that cut open plasmid and therefore we can join them together far more easily once then we have those complimentary sticky ends the DNA fragment has been cut um to create that we can then mix them together and the enzyme liase is used to stick together those sticky ends sometimes known as analing and ligas is catalyzed in the condensation reaction to form phosphodiester bonds between the sticky ends of the plasmid and the sticky ends of the isolated DNA fragment so now we've got our plasmid with the gene of Interest inserted into it we need to get that plasmid into to the host cell and that's what transformation is so the vector which is that plasmid with the recombinant um DNA next has to be inserted into the host cell because that is the cell that's going to express that Gene to create the protein and in order to do this we need to make the cell membrane of the host cell more permeable and bacteria are your typical host cells so to increase the permeability of that membrane to get the vector in the host cells are mixed with calcium ions and heat shocked which means a sudden increase in temperature and that should enable the vector to enter the host cell cytoplasm so that has now got the isolated DNA fragment into a vector and then into the host cell but we do have to check that the host is definitely taken up a recombinant plasmid so that's the next step in the process identifying trans form cells which means identifying cells that have taken up the recombinant plasmid so not all of the host cells which are usually bacteria successfully take up the recombinant plasmid and these are the three key issues that could occur maybe the recombinant plasmid just didn't get in through the cell membrane or maybe the plasmid that did get in is one that doesn't have the gene of interest in it cuz sometimes when you cut open the plasmid the plasmid sticky ends just rejoin back together and you get your original plasmid again and then sometimes the isolated DNA fragment of interests that actually loops around and it sticky and stick together to make a very very tiny plasmid and that gets inserted instead of the recombinant plasmid so that's why we have to identify the transformed cells to make sure before we grow the bacteria on mass to create that protein of interest from the gene of Interest that you're definitely growing bacteria that contain the recombinant plasmid so the way this is done is using marker genes so marker genes on the plasmid is how we identify which bacteria has successfully taken up the recombinant plasmid and the common marker genes that are used are marker genes for antibiotic resistance or coding fluorescent proteins or coding for enzymes and that's because these make it easy identify whether the bacteria has that Gene in it and therefore if it does it means it must have the plasmid within it as well so another concept that you need to be aware of within this is that Gene editing occurs and this is a precise and targeted form of genetic engineering that involves a modification of DNA at specific locations within an organism's genome and unlike traditional genetic engineering methods that typically involve the insertion of entire genes Gene editing allows for the precise insertion or deletion or replacement of DNA sequences at precise locations and this Precision is achieved using specialized tools such as crisper cast 9 which acts as a molecular scissors to cut the DNA at specific sites determined by guide RNA molecules so once the DNA is cut cellular repair mechanisms can be harnessed to introd uce desired changes to the genetic code Gene editing techniques offer unprecedented control over genetic manipulation and that allows scientists to modify genes with high accuracy and efficiency this capability has numerous applications in research and agriculture medicine and biotechnology including the development of disease resistant crops the treatment of genetic disorders and the creation of genetically modified organisms with tailored traits the gene editing holds immense promises for advancing our understanded genetics and revolutionizing various Fields by enabling this precise and targeted modification to the genome so you can also do invitro clothing which means not inside of the organism and that's is what PCR is so it's a way to amplify the DNA fragments that you have and once the DNA fragments have been isolated they need to be cloned to create large quantities and we've already gone through how you do that in Vivo in the bacteria but in vitro is PCR the polymerase Chain Reaction which is all done in an automated machine and in this PCR machine you need a thermocycler which is the machine DNA fragment that you want to make lots of copies of the enzyme DNA polymerase and it's a special version of that Tac po this Tac polymerase is taken from bacteria that um are found in Hot Springs so they're naturally adapted to survive at incredibly high temperatures so the DNA polymerase Tac polymerase has a much much higher Optimum temperature and it doesn't denat at your typical 37° C it denatures at much higher temperatures and that enables the temperature in the thermocycle to be increased to speed up the reaction with ald the enzymes primes are needed as well to attach to um the DNA fragments to enable the DNA replication to start and DNA nucleotides are needed to create your new DNA sequences so the method then the temperature is first increased to 95° C to break the hydrogen bonds between the two DNA strands so we now have two single strands of DNA and they both act as a template the temperature is then decreased to 55° C so it's cool enough that the complementary DNA primers can align opposite those complimentary bases and form hydrogen bonds to hold them in place opposite those compliment base pairs the enzyme DNA polymerase will then attach and the free floating DNA nucleotides can align opposite the complimentary base pairs DNA polymerase will then catalyze the formation of phos foder bonds between adjacent nucleotides and that's how we get the synthesis of the new DNA strand that process is then just repeated that's why it's called a thermocycler because the temperature has changed continuously in this cycle so 95 then 55 then 72° C and it's 72° c for the final stage because that's the optimum temperature for that Tac DNA polymerase so PCR is fully automated it's very efficient it's rapid you can make billions of copies of DNA within a few hours and it doesn't require living cells like the invivo did which requir bacterial cells therefore it's quicker and it's a less complex technique we're then going to move on to DNA fingerprinting which is a way to look at how closely related different individuals are based on vntrs which are variable number t tandem repeats which are found within the introns of DNA and the probability of two individuals having the same vntrs is very very low but the more closely related you are the more similar your vntrs are and that is how genetic fingerprinting works it's the analysis of vntr DNA fragments to determine genetic relationships and genetic variability within a population so here are the stages we're going to go through in genetic fingerprinting collecting your sample of DNA to analyze how you then extract just the DNA digest it separate it and then ultimately analyze your sample so collection the smallest sample of DNA can be collected for genetic finger printing and this could be from blood or body cells or hair follicules and if the sample of DNA is really small then PCR is going to be used first so you have a large quantity then we need to digest this DNA and we're going to use restriction endonucleases to cut the DNA into smaller fragments and enzymes which cut close to Target vntrs are going to be added to separate out the VN those cut up fragments the DNA samples are loaded into these tiny wells in a block of agar gel the gel is then placed in a buffer liquid with an electrical voltage applied DNA is negatively charged because of the phosphate groups there's a negative charge in that phosphate group so the DNA samples will move through the gel towards the positive end of the gel and they'll move at different speeds depending on how long the DNA fragment is so how big that particular vntr fragment is and that is how it separates out now the agar gel resistance for the moving DNA and therefore smaller pieces of DNA can move faster and further along the gel and this is how the different DNA vntrs are separated and alkaline is then added to separate the DNA from being double stranded to single stranded and the hybridization stage is when DNA probes which are short single stranded pieces of DNA which have been created to be complementary in terms of the base sequence of the VN RS those are added and the DNA probes either have a radioactive or fluorescent label so that we can then move on to the development stage but this part is called hybridization because the different DNA probes are mixed with those separated strands so you've got two different pieces of DNA joining together creating a hybrid so the development then the AAR gel will actually shrink and crack as it dries so we transfer the vntrs and DNA probes using a nylon sheet so transfer onto that and the nylon sheet can then be exposed to xrays if you've used a radioactive probe or you can expose it to UV light if it was a fluorescent probe that was used and that is then how we get this development of your bands that we can actually see the positions of so the position of the DNA bands are compared to identify genetic relationships or it could be to see the presence of a disease caused gene or to match an unknown sample of DNA from a crime scene and this is one that I actually created a long time ago we can see this was the unknown that I had to identify here were the different samples of DNA that I had and from this example I could match that the unknown was the same DNA as number three so we can see the bands are an exactly the same position and that is your analysis stage so interpreting um the DNA the results of the gel electri Rees this is your analysis stage again and if you're going to do this in a paternity test here we have the bands from the mother here are the bands from the child and any band in the child that wasn't from the mother's DNA must be from the father's and that is how you can use this in paternity tests so you'd have to match up the bands to see which bands they have in common with the different fathers to see if that particular individual could be the father you can also use micro arrays which are used to identify the genes present in an organism's genome and determine which genes are actively expressing in the cells and this technology allows researchers to study a large number of genes very rapidly at the same time so following the hybridization of DNA or an mRNA sample to the micro array which is basically a plastic dish with lots of mini um dimples in it the data are then scanned and interpreted by a computer and the processor reveals which genes are shared between species or unique to one species or absent in both it also enables the detection of gene expression patterns in specific cell types or conditions say for instance cancer cells exhibit different gene expression profiles compared to non-cancerous cells and micro arrays are used to compare gene activity between transcribed genes converting mRNA to complimentary DNA using reverse transcriptase and amplifying that complimentary DNA if necessary through PCR you then get these fluorescently labeled complimentary DNA which hybridize with the probes on the micro array and the fluorescent spots are going to indicate genes actively transcribed in the cell and with the intensity of the fluorescence correlating with the gene activity level this has been particularly useful in investigating mutations in genes associated with breast cancer shedding light on the underlying genetic mechanisms of disease so by providing the insight into gene expression patterns and activity levels these micro arrays offer an valuable information for understanding complex biological processes and facilitating advancements in genomics and biomedical research now databases are also used so Research into the genes present in various organisms and their expression generates lots of data and this data has to be stored digitally in a database and that encompasses nucleotide sequences of genomes or individual genes as well as amino acid sequences um and this field responsible for collecting processing and analyzing all of that information is known as bioinformatics and software developers actually play a crucial role in creating these systems for storing searching retrieving and analyzing this data so computer technology facilitates The Collection analysis and also internet-based access to this information notable databases include Ensemble which houses genomic data of eukariotic organisms like humans and zebra fish and mice uniprot which contains information on protein sequences and functions and the protein Data Bank which provides details on protein sequences and structures and these databases are essential resources for researchers providing valuable insights into genetics and protein biology there are also selection and retrieval tools such as the basic local alignment Search tool or Blast for short enabling researchers to compare sequences and identify similarities with existing database entries comparison between sequence Geno such as the human genome with that of a fruit fly or nematode worm has revealed evolutionary relationships and common ancestry it's been really really useful model organisms like dropa um are instrumental in studying Gene functions and developmental processes due to how easy their genetics is to work with but also how well characterized their genomes are and that information from their genome helps us understand the human gene function and disease mechanisms as well so although it's not humans we're working on we still gain valuable information to help us understand diseases Within Me diseases within humans so this vast genomic information stored in databases has so many practical applications it's been used in malaria research for example where Gene sequences have been utilized in vaccine development as well additionally databases like cft2 provide accessible information on genetic variance associated with diseases like cystic fibrosis and this helps healthc Care Professionals in diagnosis and treatment decisions then we go on to 19.2 genetic technology applied to medicine so there are advantages of using this recombinant technology genetic engineering enables the production of recombinant human proteins offering several advantages in the treatment of diseases and here are just three examples of proteins that have been created using bacteria yeast or even mamalian cell cultures um and using these bacteria yeast and mamalian cell cultures producing these human proteins are several advantages first of all these cells are uncomplicated nutritional requirements they're very very basic cells you get large quantities of the desired protein being generated that means you've got lots of this medicine available you can do this in facilities that require minimal space because these are single cells rather than having entire multicellular organisms the process can be conducted almost anywhere globally and also the protein extraction from animals or blood collection from multiple donors um would therefore minimize practical and ethical concerns you're not having to do that you're just taking it from a single cell organism that is producing it so other human proteins like the fact 8 and the adenosine Dianes are produced using similar Technologies so we've got that factor a is crucial for blood clotting and it's synthesized by genetically modifying hamster cells and by inserting the human factor 8 gene into the hamster kidney and ovary cells cultured in fermenters a constant supply of factor8 is produced and that eliminates the need for blood tations and therefore the associated infection risk and the availability of the blood adenosine deaminase is essential for Te produced using genetically modified bacteria ecoli or cell cultures derived from insect lvy this enzyme therapy has been successfully utilized for over two decades to treat skid or severe combined immuno deficiency highlighting its long-term efficacy as an alternative treatment option now genetic screening is when you would have your DNA tested to screen whether you have the gene or an alel linked to an increased likelihood of developing a disease or it could be to see if you will definitely develop a particular disease so we've got a whole list of advantages here why you might want to do this you'd get an insight into an individual's genetic makeup it might help you to prevent the development of a disease or get better management of it it could result in the early detection of certain diseases such as breast cancer where early detection is essential for increasing survival rates you can make informed decisions about your health um early diagnosis in general can help with personalized treatments for certain diseases such as Huntington's disease and cystic fibrosis it will help with preventative measures so certain diseases you won't definitely develop if you have the associated gene or alio but if you modify your lifestyle you're then going to reduce the likelihood of developing it so overall it can help to improve patient outcomes and also therefore reduce healthc care costs gene therapy is a way that we could try and treat genetic diseases as well and it involves correcting faulty genes or introducing functional ones within the genome to override the affected cells so for example severe combined amuno deficiency and inherited eye diseases have been targeted for gene therapy interventions so in skid where the deficiency is linked to Ada which is comprising the immune function gene therapy involves restoring normal Ada activity and that will then give long-term benefits in the inherited eye diseases like LCA gene therapy restores Vision by introducing functional alals of the rpe65 gene into the affected retinal cells and these examples highlight the potential of gene therapy in providing longas lasting solutions for genetic diseases therefore improving patients quality of life now so far we've only highlighted all the positives with these Technologies but there are social and ethical considerations linked to this as well so World genetic screening does empower individuals to make these informed decisions about their health it does link to some concerns around privacy discrimination and also psychological impact because in terms of privacy once you have screened yourself there's the question of well who then gets access to that information and it could be for example maybe Insurance um providers might get access and therefore they wouldn't give you health insurance anymore because they know that you are higher risk of developing a particular disease um you might get discriminated from an employer if they knew that you are likely to develop a disease later in life and also the psychological impact of knowing you have an alio that makes you at risk of developing disease that can play heavily on someone's mind so gene therapy does pose challenges related to safety efficacy and also Equitable access to treatment as well the ethical dilemmas arise regarding the manipulation of human genetic material and therefore the potential for eugenics and the allocation of resources so we do have to strike a balance between the benefits of these scientific progresses whilst also considering the ethics and the social side of the considerations as well lastly then is genetically modified organisms in agriculture so genetic engineering is one technology that could really help with the global demand for food because we could enhance the quality and productivity of farmed animals and crop plants so for example GM salmon have been developed to grow faster and more efficiently offering a potential solution to meet the increasing demand for for seafood also we've got herbicide resistant soybean varieties have been engineered to tolerate herbicides which enables Farmers to control weeds more effectively and therefore increase their crop yield we've also got insect resistant cotton varieties created by incorporating genes into BT bacteria which produce toxins lethal to certain pests and therefore it would kill the pests without needing to apply chemical pesticides therefore it's a more sustainable agriculture so this shows how you could use genetic engineering to improve food security sustainability and also economics but again there are downsides to consider link to the ethical and social implications so the use of genetically modified organisms to produce food in this way has one issue around um GM cultivation so unintended effects maybe of non-targeted organisms so maybe it does actually affect other organisms not just the crop plant of Interest it could result in maybe biodiversity loss if it causes the death of um other plants and also you might get herbicide resistant weeds and pest populations there's also concerns that this is a relatively new technology so we don't know the long-term effects of humans eating GM food and there may well be no long term effects but we actually don't know because we haven't tested it we've got social economic factors as well because these seeds are very expensive so therefore they become too expensive for poorer Farmers or poorer countries to be able to afford to buy these seeds to get these GM crops and then we've got ethical debates as well which is looking at the moral implications of manipulating living organisms and the potential consequences it might have on Animal Welfare ecosystem integrity and future Generations as well so that takes us to the end of the genetic technology topic hope you found it helpful and if you did don't forget to subscribe [Music]