Chapter 21: Flashcards
What are the three methods for producing DNA fragments?
Reverse transcriptase, gene machine restriction endonucleases.
How does reverse transcriptase produce DNA fragments?
> mRNA for a specific polypeptide is isolated from the cell. These cells should have a large amount of mRNA.
Free DNA nucleotides and reverse transcriptase are added. Reverse Transcriptase uses the mRNA as a template to produce cDNA, which is a double-stranded copy of the required gene. cDNA is complementary.
Where are restriction endonucleases found?
They naturally occur in bacteria.
What does restriction endonuclease do?
They break phosphodiester bonds and attach to the recognition site.
Describe how restriction under nucleases produces DNA fragments.
> The active site of the restriction endonucleases is complementary to specific recognition sites on DNA. >Restriction endonucleases bind to recognition sites and cut the DNA.
This forms sticky ends, revealing unpaired base sequences at either end of the fragment or plasmid.
If vector DNA is cut with the same restriction endonuclease. The vector sticky ends are complementary to the DNA fragment and are joined together by DNA ligase.
How does the gene machine work?
> First, identify the amino acid sequence and then work out the mRNA and DNA sequence.
Put the DNA sequence into the computer which checks for biosafety and biosecurity. Sections of overlapping single strands of nucleotides, called oligonucleotides can join to form the DNA sequence of a gene. >This is called recombinant DNA. PCR can be used to amplify.
What are the benefits of using the Gene Machine?
It is quick, accurate and intron free.
Once DNA fragments are produced, what must be done?
Promoter and Terminator regions must be added for transcription to occur.
What is a promoter region, and where is it added?
Promoter regions are added at the start of the DNA fragment. The promoter region is a sequence of DNA which is the binding site for RNA polymerase to enable transcriptase.
Terminator region and where is it added
Terminator region is added at the end of the DNA fragment. It causes RNA polymerase to detach. So, only one gene is copied, one at a time.
Once the DNA fragment has been modified what must occur?
Amplification of this DNA fragment through either in vivo cloning or in vitro cloning.
What happens in the in-vivo cloning process?
> Insert DNA into vector As the vector can transfer DNA into the host cell. The plasmid is then cut with the same restriction endonucleases, which creates the same sticky ends. DNA fragments and plasmid sticky ends are complementary; DNA ligase then catalyses phosphodiester bonds between nucleotides.
Transferring recombinant DNA into host cells: the vector is next inserted into her cells, where the gene will be expressed. Cell membrane of the host cell must be more permeable, so is mixed with calcium 2 plus ions and heat shocked to enable the vector to enter the cytoplasm.
Identify transformed host cells: Fluorescent gene is inserted into the plasmid. The DNA fragment inserted disrupts the gene for fluorescent plasmids. Recombinant= No colour under UV light.
Fermenter is used to grow more copies of hotels which have been identified as containing plasmids.
What equipment is needed for in vitro cloning?
Thermocycler, TAC polymerase (polymerase from bacteria in Hot Springs), DNA nucleotides and primers (which are short sequences that have bases complementary to DNA fragment.)
What occurs In in vitro cloning?
> The temperature needs to rise to 95° to break hydrogen bonds between the two strands.
The temperature then needs to be cooled to 55° so primers can attach as complementary bases at the end of the fragment.
The temperature must be raised to 75°, which is optimum for TAQ polymers, which allows DNA fragments to attach to complimentary free nucleotides and make new strands next to each strand. Primers add nucleotides in sequence on both strands
How are DNA probes formed?
Locate DNA base sequence must be known to create the DNA probe. Fragment of DNA produced using gene machine and can be amplified with PCR. A radioactive fluorescent label is added.
What occurs in hybridization?
> Denaturing the DNA needs to be single-stranded, so it’s heated, and the heat hydrogen bonds break, forming a straight single-stranded molecule.
Single-stranded DNA and probe are added. This is then cooled, and any complementary sequences can align and join. Also known as annealing. DNA is washed so unbound probes are washed away.
What is genetic counselling?
A type of social work allows patients to make informed choices
What occurs in genetic fingerprinting?
> DNA is collected from blood etc and is amplified with PCR if needed.
Then, restriction endonucleases are added. They cut into the DNA fragment just before the VNTRs.
Then, place the sample into wells in the Agar Jelly.
Then, an electrical voltage is applied, and the DNA, which is negatively charged, will move to the positive end of the gel.
Gel provides resistance, which means smaller VNTRS will move further, thus separating them.
Then an alkali is added and add DNA probes which are complementary they are radioactively or fluorescently labelled. They then bind and we washed to remove any unbound.
Suggest one reason why DNA replication stops in the polymerase chain
reaction.
Primer or nucleotides run out
Scientists have used the RT-PCR method to detect the presence of
different RNA viruses in patients suffering from respiratory diseases.
The scientists produced a variety of primers for this procedure.
Explain why.
mass of polypeptides
Charge;
Describe the roles of one of the named types of enzymes used to insert DNA
fragments into plasmids.
DNA ligase - joins DNA to plasmid/vector
Suggest and explain how delayed insertion of the GH gene could produce
offspring of transgenic fish without the desired characteristic.
- Cell division has occurred (before gene added);
- gametes do not receive the gene;
Does reverse transcriptase, gene machine and restriction endonuclease produce recombinant DNA
Reverse transcriptase, gene machines, and restriction endonucleases do not on their own produce recombinant DNA -> part of the process
So When Does Recombinant DNA Actually Form?
> A gene (DNA fragment) (from reverse transcriptase, gene machine, or cut out by restriction enzyme)
is inserted into a different DNA molecule (usually a plasmid or vector)
and the two pieces are joined by DNA ligase.
What is the job of reverse transcriptase, gene machine and restriction endonuclease
Isolate the Gene You Want
What does reverse transcriptase do?
Converts mRNA → complementary DNA (cDNA)
Explain what occurs in RT-PCR.
PCR only occurs on DNA, so RNA -> must be converted to cDNA first
Suggest how single-stranded cDNA could prevent transcription of the P34
- The single-stranded cDNA produced would be complementary to the mRNA transcript of the P34 gene.
- By binding to the P34 mRNA, the single-stranded cDNA could prevent the mRNA from being translated into the P34 protein.
The geneticist concluded it would be faster to create the HGH gene using a
gene machine than by using reverse transcriptase to convert mRNA for
HGH into cDNA.
Suggest why the geneticist reached this conclusion.
Faster to use gene machine than all the enzyme-catalysed reactions
(involving reverse transcriptase);
What would the scientists have inserted into the plasmid along with the
spider gene to ensure that the spider gene was only expressed in the silk
glands of the silkworms?
Promoter Region
