8 Recombinant DNA tech Flashcards
recombinant dna technology
Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism.
how is it possible that the dna of one organism is accepted by another species which functions normally when it is transferred?
the genetic code is the same in all organisms
it is universal and can be used by all living organisms
what are the stages of the process of making a protein using the dna technology of gene transfer and cloning?
- isolation- of the dna fragments that have the gene for the desired protein
- insertion- of the dna fragment into a vector
- transformation- that is, the transfer of dna into suitable host cells
- identification- of the host cells that have successfully taken up the gene by use of gene markers
- growth/cloning- of the population of host cells
what are the several methods of producing dna fragments?
- conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
- using restriction endonucleases to cut a fragment containing the desired gene from DNA
- creating the gene in a ‘gene machine’.
using reverse transcriptase
- a cell that readily produces the protein is selected (e.g. the B-cells of the islets of Langerhans from the pancreas are used to insulin)
- these cells have large quantities of the relevant mRNA, which is therefore more easily extracted
- reverse transcriptase is then used to make DNA from RNA. this DNA is known as complementary DNA (cDNA) as it is made up of the nucleotides that are complementary to the mRNA
- to make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. this double strand of DNA is the required gene
using restriction endonucleases
RE are enzymes that cut up viral dna
many kinds of RE, each one cuts a dna double strand at a specific sequence
sometimes, this cut occurs two opposite base pairs, leaving two straight edges known as blunt ends
other RE cut dna in a staggered fashion, leaving an uneven cut in which each strand of the dna has exposed, unpaired bases
the gene machine
- the desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce. the AA sequence of this protein is determined. from this, the mRNA codons are looked up and the complementary dna triplets are worked out.
- the desired sequence of nucleotide bases for the gene is fed into a computer
- the sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements
- the computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene
- in an automated process, each of the oligonucleotides is assembled by adding one at a time in the required sequence
- the oligonucleotides are joined together to make a gene. this gene doesn’t have introns. the gene is replicated using the polymerase chain reaction.
- the polymerase chain reaction also constructs the complementary stand of nucleotides to make the required double stranded gene. it then multiplies this gene many times to give numerous copies.
- using sticky ends the gene can then be inserted into a bacterial plasmid. this acts as a vector for the gene allowing it to be stored, clones or transferred to other organisms.
- the genes are checked using standard sequencing techniques and those with errors are rejected
advantages of the gene machine
any sequence of nucleotides can be produced, in a very short time and with great accuracy
these artificial genes are also free of introns and other non-coding dna, so can be transcribed and translated by prokaryotic cells
in what ways can fragments of dna be cloned so there is a sufficient quantity for medical and commercial use?
in vivo- by transferring the fragments to a host cell using a vector
in vitro- using the polymerase chain reaction
importance of sticky ends
sequences of dna that are cut by restriction endonucleases are called recognition sites
if the recognition site is cut in a staggered fashion, the cut ends of the dna double strand are left with a single strand at which is a few nucleotide bases long.
the nucleotides on the single strand at one side are complementary to those at the other side
if the same RE is used to cut dna, then all the fragments produced will have ends complementary to one another.
meaning that the single-stranded end of any one fragment can be joined to any other fragment
once the complementary bases of two sticky ends have paired up, an enzyme called dna ligase is used to bind the phosphate-sugar framework of the two sections of dna and so unite them as one
sticky ends are important, provided the same RE is used, we can combine the dna of one organism with that of any other organism
preparing the dna fragment for insertion- in vivo cloning
- the preparation of the dna fragment involves the addition of extra lengths of dna
- for the transcription of any gene to take place, the enzyme that synthesises mRNA (RNA polymerase) must attach to the dna near a gene
- the binding site for RNA polymerase is a region of dna, known as a promoter
- the nucleotide bases of the promoter attach both RNA polymerase and transcription factors and so begin the process of transcription
- another region releases RNA polymerase and ends transcription
- this region of dna is called a terminator
insertion of dna fragment into a vector- in vivo cloning
- once the dna fragment has been prepared for insertion, it is joint to a carrying unit, known as a vector
- this vector is used to transport the dna into the host cell
- there are diff types of vector but the most commonly used is the plasmid
- plasmids are circular lengths of dna, found in bacteria which are separate from the main bacterial dna
- plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop
- the RE used is the same one that cut out the dna fragment, ensuring that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the dna fragment
- when the dna fragments are mixed with the opened-up plasmids, they become incorporated into them
- where they are incorporated, the join is made permanent using the enzyme dna ligase
introduction of dna into host cells- in vivo cloning
- once the dna has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells. this process is called transformation
- transformation involves the plasmids and bacterial cells being mixed tog in a medium containing calcium ions
- the calcium ions, and changes in temp, make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm
- however, not all the bacterial cells will possess the dna fragments with the desired gene for the desired protein
why don’t all the bacterial cells possess the dna fragments with the gene for the desired protein?- in vivo cloning
- only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together
- some plasmids will have closed up again without incorporating the dna fragment
- sometimes the dna fragment ends join together to form its own plasmid
gene markers- in vivo cloning
there are a number of different ways of using marker genes to identify whether a gene has been taken up by bacterial cells
they all involve using a second, separate gene on the plasmid
this second gene is easily identifiable, e.g. it may be resistant to an antibiotic, make a fluorescent protein that is easily seen, or produce an enzyme whose action can be identified
antibiotic-resistance marker genes- in vivo cloning
to identify those cells with plasmids that have taken up the new gene we use a technique called replica plating
this process uses the other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene
as this gene has been cut, it will no longer be able to produce the enzyme that breaks down a specific antibiotic, so we can therefore identify these bacteria by growing them on a culture that contains the antibiotic
the treatment with the antibiotic will destroy the cells that contain the required gene, so by using a technique called replica plating, it is possible to identify living colonies of bacteria containing the required gene
fluorescent markers- in vivo cloning
view cells under microscope
retain those that do not fluoresce
dna probes
a short, single-stranded length of dna that has some sort of label attached that makes it easily identifiable
the two most commonly used probes are:
-radioactively labelled probes
-fluorescently labelled probes
radioactively labelled probes
made up of nucleotides with the isotope 32P
the probe is identified using an x ray film that is exposed by radioactivity
fluorescently labelled probes
emit light (fluoresce) under certain conditions, e.g. when the probe has bound to the target dna sequence
in what way are dna probes used to identify particular alleles of genes?
- a dna probe is made that has base sequences that are complementary to part of the base sequence of the dna that makes up the allele of the gene that we want to find
- the double stranded dna that is being tested is treated to separate its two strands
- the separated dna strands are mixed with the probe, which binds to the complementary base sequence on one of the strands. this is known as dna hybridisation
- the site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits
dna hybridisation
- takes place when a section of dna or rna is combined with a single-stranded section of dna which has complementary bases
- before hybridisation can take place, the two strands of the dna molecule must be separated
- this is achieved by heating dna until its double strand separates into its two complementary single strands (denaturation)
- when cooled, the complementary bases on each strand recombine (anneal) with each other to reform the original double strand
- given sufficient rime, all strands in a mixture of dna will pair up with their partners
- if, however, other complementary sections of dna are present in the mixture as the dna cools, these are just as likely to anneal with one of the separated dna strands as the two strands are with one another
locating specific alleles of genes purpose
- using dna probes and dna hybridisation, it is possible to locate a specific allele of a gene
- e.g. we may wish to determine whether someone possesses a mutant allele that causes a particular genetic disorder
process of locating specific alleles of genes
- first determine the sequence of nucleotide bases the mutant allele we are trying to locate. this can be achieved using dna sequencing techniques. however, we now have extensive genetic libraries that store the base sequences of most genetic diseases and so we can simply refer to these to obtain the sequence
- a fragment of dna is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate
- multiple copies of our dna probe are formed using the polymerase chain reaction
- a dna probe is made by attaching a marker, e.g. a fluorescent dye, to the dna fragment
- dna from the person suspected of having the mutant allele we want to locate is heated to separate its two strands
- the separated strands are cooled in a mixture containing many of our dna probes
- if the dna contains the mutant allele, one of our probes is likely to bind to it as the probe has base sequences that are exactly complementary to those on the mutant allele
- the dna is washed clean of any unattached probes
- the remaining hybridised dna will now be fluorescently labelled with the dye attached to the probe
- the dye is detected by shining light onto the fragments causing the dye to fluoresce which can be seen using a special microscope
