Recombinant DNA technology

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  • Created by: Hindleyc
  • Created on: 19-04-19 16:13
What does recombinant DNA technology allow
genes to be manipulated, altered and transferred from organism to organism- even to transform DNA itself
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What have techniques allowed us/ enabled us to understand better
how organisms work and to design and to design new industrial processes and medical applications
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What do a number of human diseases result from
individuals being unable to produce for themselves various metabolic chemicals (many are proteins eg insulin)
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they are therefore the product of
a specific length of DNA that is the product of a gene
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Treatements of such deficiencies previously involved
extracting chemicals from a human or animal donor and introducing it into the patient
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What do this present
problems such as rejection by IS and risk of infection - cost also considerable
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So what are there advantages in
producing large quantities of 'pure' proteins from other sources
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As a result what has happened
techniques have been developed to isolate genes , clone them and transfer them into microorganisms
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What are these microorganisms then
grown to provide a 'factory' for the continuous production of a desired protein
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So what is recombinant DNA
the DNA of 2 different organisms that has been combined in this way
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What is the resulting organism
transgenic or genetically modified organism (GMO)
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But how is the DNA of 1 organism accepted by a different species and function normally when it is transferred?
Genetic code is the same in all organisms - universal and can be used by ALL living organisms
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What does this explain
Why coded information on the transferred DNA can be interpreted
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Also what is making proteins
universal as mechanisms of transcription and translation essentially the same in all living organisms
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So as a result what can the transferred DNA be
transcribed and translated within the cells of the recipient (transgenic organism) and the proteins it codes for can be manufactured in the same way as they would be within the donor organism (all indirect evidence for evolution)
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What are the stages of the process of making a protein using the DNA technology of a gene transfer and cloning
Isolation of the DNA fragments that have the gene for the desired protein, insertion of the DNA fragments into a vector, transformation, that is, the transfer of DNA into a suitable host cell,
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then
identification of the host cells that have successfully taken up the gene by use of gene markers then growth/cloning of the population of host cells
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Before a gene can be transplanted what must it first be
identified and isolated from the rest of the DNA
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Given that required gene may consist of a sequence of a few hundred bases amongst the many millions in human DNA what is it not
a small feat
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What are the several methods of producing DNA fragments
Conversion of mRNA to cDNA using reverse transcriptase, Using restriction endonuclease to cut fragments containing the desired gene from DNA, creating the gene machine usually based on a known protein structure
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What are retroviruses
group of viruses of which the best known is HIV
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What is the coded genetic information of retroviruses
is In the form of RNA
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However is a host cell what are they able to do
synthesise DNA from their RNA using an enzyme called reverse transcriptase
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What does this do
Catalyse production of DNA from RNA (reverse of transcription of RNA from DNA)
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So what is the process
Cell that readily produces the protein is selected eg B cells of IOL from pancreases used to make insulin
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why
these cells have large quantities of the relevant mRNA which is therefore more easily extracted
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What is then used
reverse transcriptase to make DNA from RNA
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What is this DNA known as
complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA
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To make the other strand of DNA what happens
enzyme DNA polymerase used to build up the complementary nucleotides on the cDNA template
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And what is this double strand of DNA
the required gene :)
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Using RE what do all organisms use
defensive measures against pathogens eg Bacteria are frequently infected by viruses that inject their DNA into them in order to take over the cell
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So what do some bacteria defend themselves
by producing enzymes that cut up the viral DNA= restriction endonucleases
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What does each RE do
recognises and cuts DNA at a specific sequence of bases
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What do these sequences occur in
DNA of all species of all organisms but not in the same places
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What are there many types of and what do they do
R.E, each cuts a DNA double strand at a specific sequence of bases called a recognition sequence
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sometimes where does this cut occur
between 2 opposite base pairs
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What does this leave
2 straight edges known as blunt ends eg one RE cuts middle of recognition sequence GTTAAC
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What do other RE cut DNA in
a staggered fashion
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What does this
an uneven cut in which each strand of the DNA has exposed unpaired bases eg RE recognises that recognises a 6 bp AAGCTT
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What can the seq of unpaired bases that remain read
2 seq are opposites of one another- they are a palindrome
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What is the recognition sequence therefore referred to as
6 base pair palindromic seq
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What is this feature typical of
the way RE cut DNA to leave sticky ends (important)
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Staggered cut=
sticky ends
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Straight cut
= blunt ends
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What does the gene machine mean that it is now possible
to manufacture genes in a lab in the following manner
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step 1
desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce
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Then
AA sequence of this protein is determined, from this, mRNA codons are looked up and the complementary DNA triplets are worked out
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Then
the desired seq of nucleotide bases for the gene is fed into a computer
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What Is the seq checked for
biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements
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What does the computer design
a series of small overlapping single strands of nucleotides called oligonucleotides which can be assembled into the desired genes
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In an automated process what are each of the oligonucleotides
assembled by adding one nucleotide at a time in the required sequence
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What are the olingonucleotides then
joined together to make a gene
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what doesn't this gene have
introns or other non-coding DNA
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How is the gene then replicated
using the polymerase chain reaction
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What does the PCR also
constructs the complementary strand of nucleotides to make the required double stranded gene and then multiplies this gene many times to give numerous copies
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How is the gene then inserted
using sticky ends, gene can be inserted into a bacterial plasmid
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What does this act as
a vector for the gene allowing it to be stored, cloned or transferred to other organisms in the future
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How are the genes checked and those with errors?
using standard sequencing techniques and those with errors are rejected
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What is the advantage to this process
any sequences of nucleotides can be produced in a very short time (as little as 10 days) and with great accuracy
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further advantage
is that these artificial genes are also free of introns and other 'non-coding' DNA so can be transcribed and translated by prokaryotic cells
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brief summary
desired nucleotide seq fed into a computer, synthesis of oligonucleotide, assembly or gene- olingonucleotides are overlapped then joined together and made double-stranded using PCR then gene cloning as gene is inserted into a bacterial plasmid
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then
genes are seq and those with errors are rejected and gene is usually delivered incorporated into plasmid
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Card 5

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