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37 Cards in this Set
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- Back
- 3rd side (hint)
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why do we manipulate genes |
manipulating,altering and transfering genes from one orgaanism to another enables us to: -understand better how organisms work -design new industrial processes and medical applications |
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what is recombinant DNA technology |
-the processes by which genes are manipulated altered or transferred from organism to organism also as genetic engineering |
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what is the genetic code |
-the genetic code is universal -so when a piece of DNA from one organism is placed in another,it will stll code for the same polypeptides and proteins |
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describe recombinant DNA |
-DNA from two different sources that has been joined together |
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describe transgenic organisms |
-organisms that have has DNA from another individual often from another species inserted into their genome -transgenic organisms therefore contain recombinant DNA |
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why do biologists tend to work with fragments of DNA |
-they can produce these fragments in 3 different ways 1. use of reverse transcriptase to convert mRNAA to cDNA 2. use of restriction endonuclease to cut DNA fragments out of a large DNA molecule 3.creatng the gene in a gene machine |
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give an overview of gene cloning |
isolation of dna fragments insertion transformation selection culturing |
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describe isolation |
-removing the DNA from its source -a detergent breaks down the cell membranes and cell walls -once DNA is free the surrounding proteins are removed using digestive enzymes -next identify the specific gene and separate it from the rest of the organisms DNA |
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how do we isolate the gene |
-reverse transcriptase -restriction endonuclease |
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describe reverse trnscriptase |
-the cytoplasm of a specialised cell contains mRNA -this mRNA has been transcribed from the genes in the cells nucleus -it is often easier to extract mRNA from the cytoplasm of a cellto find the gene from which it was transcribed -reverse transcriptase is an enzyme which can be used to make a DNA copy from a mRNA molecule. -it therefore has no introns and can be translated by prokaryotic bacteria
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describe mRNA encoding in insulin |
-will be found in high concentrations in the cytoplasm of beta cells in the islets of langerhans but in no other cell. -molecular biologists can extract this mRNA and make a copy of DNA from it -DNA that is formed in this way is called complementary DNA or cNDA |
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how do you produce DNA from cutting DNA |
-using restricition enzymes to cut around gene -there are thousands of different restriction enzymes each cutting in a different sequence -usually palindromic sequences |
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describe restriction endonuclease |
-they are enzymes which break bonds in sugar phosphate backbone of the DNA molecule -the enzymes only breaks a particular sequence of bases -these sequences are called restriction bases -they can cut DNA in two ways 1.simple cut across the middle producing blunt ends 2.some produce fragments with short single stranded overhead.these sticky ends can form base pairs with other complemntary sequences |
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describe the gene machine |
-if the primary structure of a protein is known its possible to synthesise the gene required to produce a protein -the amino acid sequence required is entered into a computer. the triplet code for each amino acid is known so the DNA sequence that will produce that protein is worked out -the computer then controls the machine and required the DNA sequence is made |
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describe in vivo |
in life the gene is introduced to a cell and then is copied as the cell divides |
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describe in vitro |
in glass DNA is copied many times over by polymerase chain rection using per machine |
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describe vectors |
-a length of DNA that carries the gene of intrest into the host cell -it has to be big enough to contain the gene -circular-easier to br broken down -it contains control sequences -it will contain marker genes so that the gene can be identified -example is plasmids |
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describe plasmids |
-circular DNA found in bacteria -foreign DNA can easily be inserted in to them using restriction enzymes and DNA ligase |
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how to prepare the DNA fragment for instertion |
-a promoater region is attached to the DNA at the start of the gene. this is a binding site for RNA polymerase to start transcription to makea protein -a terminator region is added tot he other end of the fragment to stop transcription by releasing RNA polymerase |
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descibe insertion |
-the joining of your DNA strands with the plasmid -DNA ligase joins nucleotides in a DNA strand -two complementary sticky ends will anneal by weakhydrogen bonds -DNA ligase completes the backbone by forming covalent bonds -the DNA of two different organisms that have been combined in this way is known as recomninant DNA |
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describe transformation |
-incorporating the gene into a living cell so that it can be expressed -vectors are large so cells have to be made permeable -there are many different ways of transferring a vector into a cell these include -heat shock -electroporation -viruses |
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describe selection |
-not all vectors will be taken up by the cell -the ones with the desired vector in need to be identified |
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describe genetic makers |
-used to identify cells that have taken up the vector -usually a genethat gives resistance to antibiotics |
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describe replica plating |
-used to identify cells that have taken up the desired plasmid -cells with the desired plasmid are not resistant to ampicillin -a replica agar plate is made of the original colonies and the bacteria that contain the foregin gene -once the colonies of cells containig the correct desired plasmid vector hve been identifed the appropriate colonies on the master plate can be selected and grown on another plate |
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what are some other genetic markers |
-fluorscent markers and enzyme markers - a gene from jellyfish that produces a protein called green fluorsent protein (GFP) can be used. the desired gene is transplanted into the centre of the GFP gene. the cells that have not taken up the desired gene will fluoresce -enzyme markers involves transplanting the desired gene into the centre of a gene coding for lactase. lactase will turn a particular colourless substance blue, therefore cells that have taken up the desired gene will not turn this substance blue. |
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describe culturing |
-gene products are required on a large scale so they are cultured in a fermenter -as the bacteria grows the gene within the plasmids synthesises its protein -this product accumulates and is extracted. |
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what is complementary dna |
dna that is amde from mrna in a process that is the reverse of normal transcription |
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describe restriction endonuclease |
enzyme used to cut DNA molecules |
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descrieb reverse transcriptase |
enzyme involved in copying an mrna code into a dna molecule |
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what are sticky ends |
ends of dna fragments which haave been cut with a restriction enzyme and whcih can be joined to anoter similarly cut piece o DNA. |
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describe in vitro cloning |
-artificial DNA replication -PCR can amplify DNA samples as smaal as a single molecule -developed by kary mullis in 1993 |
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describe the process of in vitro cloning |
1. the DNA sample 4 nucleotides and DNA polymerase are added together in a thermocycler 2.double helix strands are separated by heating at 95 degrees (to break hydrogen bonds between bases) 3.DNA primers are added (a shor length of DNA sequence).the DNA is cooled to 55 degrees to allow the primers to anneal 4.DNA polymerase (taq polymerase) extends primers and completes DNA synthesisat 72 degrees (optimum temperature for enzyme) 5. this process is repeated from step 2. normaally PCR runs for 20-30 cycles. |
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what makes taq polymerase different from many other DNA polymerase |
it comes from bacteria that live in hot springs so it can withstand high temperatures without denaturing (thermostable) |
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wht are the the advantages of in vivo |
-can be used to introduce a gene into another organism -little risk of conamination -very accurate -cuts out specific genes -produces transformed bacteria that can be used to produce large quantities of product. |
in life |
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what are the advantages of in vitro |
-very rapid -dos not require living cells -can be fully automated -product can be used for further studies such as cloning and electrophoresis. |
in glass |
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what are the benefits of recombinant DNA technology |
-microorganisms can be modified to produce a range of substances -genetically modified plants can be transformed to produce a specific substance in a particular organ of the plant or they can be manufatured to produce antigens -genetically modified crops can be engineered to have financial and environmental advantages such as making plants more likely to survivr harsh conditionssuch as drought -genetically modified crops can help prevent certain diseases -genetically modifed animals are able to produce expensive drugs -replacing defective genes might cure some genetic disorders such as cystic fibrosis -genetic fingerprinting can be used in forensic science |
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what are the risks of using recombianant DNA technology |
-it is impossible to predict with complete accuracy wht the ecological consequences of this will be -a recombiant gene ay be passed to other organisms - any manipulation in DNA will have consquences for the metabollic pathways within the cell. -genetically modifed bacteria often have an antibiotic resistance marker genes -we can not be certain of the long term consequences -what might be the finacial consequences of developing plants and animals to grow in new regions -is the finacial cost of thiis technology justified. |
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