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Topic 8
Creating DNA fragments
| Question | Answer |
|---|---|
| What is Recimbinant DNA technology | The combining of different organisms DNA which could enable scientists to manipulate and alter genes to improve industrial processes and medical treatment |
| The first step in this techonology is to produce or | Isolate the fragments of a DNA to be recombined with another piece of DNA |
| 3 steps to create fragments of DNA | Reverse transcription Restriction endonucleases Gene machine |
| Process of reverse transcription 1 | This enzyme makes DNA copies form mRNA |
| 2 | Naturally occurs in viruses such as HIV |
| 3 | A cell that naturally produces the protien of interest is selected |
| 4 | These cells should have large amounts of mRNA for the protein |
| 5 | The reverse transcriptase enzyme joins the DNA nucelotides with complementary bases to the mRNA sequnece |
| 6 | Single stranded DNA is made |
| 7 | To make this DNA fragment double stranded the enzyme DNA polymerase is used |
| Advantage of reverse transcription | The cDNA is intron free because it is bases on the mRNA template |
| What are restriction endonucleases | These are enzymes that cut up DNA These naturally occur in bacteria as a defense mechanism |
| Recognition sequences is described through when | Restriction enzymes have an active site complementary in shape to a range of differnet DNA base sequences described as recognition sequences |
| Enzyme cuts DNA at a specific location | |
| Some enzymes cut at the same location in the double strand and create a blunt end. | |
| Other enzymes cut to create staggered ends and exposed DNA bases | |
| They are known as sticky ends as they have the ability to join to DNA with complementory base pairs | |
| DNA fragments can be created | In a lab using computerised machines |
| Scientists first examine the protein of interest to identify the amino acid sequence and from that work out what mRNA and DNA sequence would be | |
| DNA sequence entered into a computer which checks for biosafety and biosecurity that the DNA being created is safe and ethical to produce | |
| The computer can create small sections of overlapping single strands of nucelotides that make up the gene called oligonucleotides | |
| Oligonucleotides can then be joined to create the DNA for the entire gene | |
| PCR can be used to amplify the quantity and to make the double strand | |
| Advantages of reverse transcriptase | mRNA present in cell is from activley transcribed genes so lots of mRNA of interest is available to make cDNA |
| Disadvantage of reverse transcriptase | More steps so more time consuming and technically more difficult |
| Advantages of Restriction endonuclease | Sticky ends on DNA fragment make it easier to make recombinant DNA |
| Disadvantages of restriction endonuclease | Still contains introns |
| Advantages of gene machines | Can design exact DNA fragment you want with sticky ends labels and preferential codons |
| Disadvantages of gene machines | Need to know the sequence of amino acids or bases |
Created by:
yHya