AQA BIOL5 Biology Unit 5 Exam - 22nd June 2011

Original post by angel1992

hey people, jus wanted to know bacteria cant splice out introns so is the gene that is inserted already spliced, this would mean it would be produced be reverse transcriptase right? because this already has spliced out the introns, and if so how is this gene produced by reverse transcriptase inserted into a plasmid if its isolated by reverse transcriptase rather than restriction endonuclease??

also does this mean that in vivo gene cloning always involves reverse transcriptase?

Correct - Bacteria do not possess the enzymes for splicing, hence the gene that is inserted is already spliced.

The easiest way to get a spliced gene is from the mRNA of the cell, as long as it is produced in sufficient amounts (otherwise will be quiet hard to purify).

mRNA is converted to DNA with reverse transcriptase (which makes a single strand of DNA from mRNA). ssDNA is converted to ds DNA using DNA polymerase. The ds DNA is then cut with restriction enzymes and inserted into the vector.

Each exon codes for one polypeptide chain, sometimes it is sufficient to clone just one exon - in this case the DNA is directly extracted from the cell, cut with restriction enzymes and used for ligation.

Not all genes will have introns. Introns are more common in genes which code for things like antibodies - in antibody fragments, the constant regions remains the same over time, but the variable region changes depending on the antigen. Hence it is important that the cell be able to mix and match different variable regions to the constant region - this is where introns/exons are needed.

Reply 1062

i dont understand why primers are needed in pcr, when in dna replication dna polymerase can just join up bases to the chain without primers?

Reply 1063

tehsponge

11

Original post by Cyanohydrin

'How Science Works is a load of cack used to fill up space and piss people off. Discuss'

I would just rewrite the shrew question from last year :tongue:

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Sparkly-Star

OP

13

There are tons of animations related to unit 5 on this site, http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter10/animation__action_potentials_and_muscle_contraction.html you do need to do an advanced search on Google to find the topics though.

(edited 12 years ago)

Reply 1065

appleschnapps

I dream of getting a question about the transport and uses of oxygen, though something like cell specialisation wouldn't be too bad.

Reply 1066

Original post by flowerscat

Correct - Bacteria do not possess the enzymes for splicing, hence the gene that is inserted is already spliced.

The easiest way to get a spliced gene is from the mRNA of the cell, as long as it is produced in sufficient amounts (otherwise will be quiet hard to purify).

mRNA is converted to DNA with reverse transcriptase (which makes a single strand of DNA from mRNA). ssDNA is converted to ds DNA using DNA polymerase. The ds DNA is then cut with restriction enzymes and inserted into the vector.

Each exon codes for one polypeptide chain, sometimes it is sufficient to clone just one exon - in this case the DNA is directly extracted from the cell, cut with restriction enzymes and used for ligation.

Not all genes will have introns. Introns are more common in genes which code for things like antibodies - in antibody fragments, the constant regions remains the same over time, but the variable region changes depending on the antigen. Hence it is important that the cell be able to mix and match different variable regions to the constant region - this is where introns/exons are needed.

hey thanx that was extermly helpful, one thing though you know with the doubl stranded dna, this is a gene, so if you used restriction enzymes on it wouldnt you be cutting up the gene?

Reply 1067

Original post by angel1992

hey also one more question with the antibiotic markers why dont you just test whether the plasmids have resistance for tetracycline straight away without hving to do the ampicilllin thing?

because the efficiency of transformation (insertion of vector into bacteria) is not 100% - there will be some cells that do not take up plasmid at all. You need to get rid of these cells first by growing on ampicillin medium - all cells that have taken up the plasmid will survive.

In the case of GFP or enzyme markers, a single step selection is possible - after transformation, grow the cells on an ampicillin containing medium - cells that have not taken up plasmid will die, those that do not carry the gene of interest will glow green and those that have vectors with the gene of interest will survive but not glow green.
Same principle with enzyme markers - the gene coding for the enzyme will metabolise a substrate in the medium and turn it blue.

Reply 1068

Original post by diskohuelga

Anyone want to tell me the actual importance of T-tubules? What are they there for?

The calcium ions released from the sacroplasmic reticulum diffuse down the t-tubules, spreading rapidly down the muscles. This ensures that the entire muscle contracts together.

Reply 1069

i dont understand why dna polyemrase chain reactions are useful with small samples of dna becase in say a drop of blood, you still have probably millions of cells in them right?? so how is it useful?

Reply 1070

xelaman

Original post by angel1992

i dont understand why dna polyemrase chain reactions are useful with small samples of dna becase in say a drop of blood, you still have probably millions of cells in them right?? so how is it useful?

The main constituent of blood is plasma-but i guess like any biological test you want to repeat it lots of times, so that you can work out an average and identify any anomalies making your results more reliable. :smile:

Reply 1071

Original post by angel1992

i dont understand why primers are needed in pcr, when in dna replication dna polymerase can just join up bases to the chain without primers?

Both Taq polymerase (PCR) and DNA polymerase (replication) need primers.

During replication the first two bases are RNA, made by the enzyme primase, which acts as a "primer" for DNA polymerase to bind to. These are later removed when the DNA strands are joined up.

Its just not mentioned in your syllabus :rolleyes:

Reply 1072

Cyanohydrin

13

Original post by flowerscat

The calcium ions released from the sacroplasmic reticulum diffuse down the t-tubules, spreading rapidly down the muscles. This ensures that the entire muscle contracts together.

??

the t-tubules connect the membrane to a protein complex

this protein complex is connected to a sarcoplasmic reticulum

when an action potential passes down the t-tubule and reaches the protein complex it stiumulates the protein complex to signal the release of calcium ions from the sarcoplasmic reticulum

Reply 1073

Original post by angel1992

hey thanx that was extermly helpful, one thing though you know with the doubl stranded dna, this is a gene, so if you used restriction enzymes on it wouldnt you be cutting up the gene?

you would be cutting the "ends" of the gene - does that make sense? So the sequence of the gene - at least the section coding for the polypeptide chain are preserved intact.

Reply 1074

Original post by angel1992

i dont understand why dna polyemrase chain reactions are useful with small samples of dna becase in say a drop of blood, you still have probably millions of cells in them right?? so how is it useful?

Not all the DNA will be of good quality - only some of it will be suitable for analysis. So you would want to use PCR to salvage the "good" DNA - then you don't need to worry if you mess up your restriction digest or sequencing down the line :-)

Reply 1075

tehsponge

11

Original post by flowerscat

Not all the DNA will be of good quality - only some of it will be suitable for analysis. So you would want to use PCR to salvage the "good" DNA - then you don't need to worry if you mess up your restriction digest or sequencing down the line :-)

True, but PCR has a high chance of error in the DNA replication involved in it, which needs to be considered too

Reply 1076