AQA BIOL5 Biology Unit 5 Exam - 22nd June 2011

Only in tRNA, between bases to give that clover shape. And in double stranded RNA.

ok so where to start right
two ways to make dna fragments

reverse transcriptase- is an enzyme converting mrna into dna
useful because introns so can be used to transform bacteria only, which can then produce the proetins( bacteria cant slice)

restriction endonucleases- these are made by bacteria to cut up viral dna
they cut at recogntion sites which ususally have palindromic sequences
sticky ends are produced because of the bases being revealed, and they are better than blunt ends as they are more specific
restriction endonucleases can be used in vivo cloning to cut out a gene ( which has these recognition sites either side) and stuck into a plasmid cut once using the same restriction endonuclease

these plasmids can used as vectors to insert into host cells
note that if you want to transform a bacteria you usually use reverse transcriptase as well as restriction endonuclease but if you want to transform an animal or a plant you dont have to use reverse trancriptase becuase these organsims can splice out introns



in vivo gene cloning clones directly and isnt done in living cells, you cant transform organisms in this way, as organisms are transformed through vivo cloning as the recombinant dna is replicated as part of their development





in vitro is better as more dna is made in a shorter space of time, and also it doesnt need living cells so reduceds the need for growth mediums and culturing etc, but as its less specific it means that contaminant dna is also amplified,in addtion its less accurate because gene cloning for example conducts less errors, as its in living tissue so although mutations occur they are rare, while in vitro clonning isnt in lving tissue and so its artificial dna cloning and more errors are made


gene therapy can treat cystic fibrosis as well as scid, by the use of adeno viruses, retrovirsues, or liposomes, and could either use somatic cell therapy or germ line therapy where germ line is negative because of the use of fertilised eggs and manipulating them , while its positive as it prevents immune reponses and is longer lived in that repeated treatement arent needed and isnt passed on to future regenerations. aduly Stem cells can also be used here, in somatic cell therapy, as they prevent repeated treatments being needed but it doesnt eliminate the fact that it is passed onto future geneartions..

sequencing a gene
sanger method/chain termination method
restriction mapping- which aids sequencing as enables larger genes to be sequenced

locating a gene- this is done after the sequencing method- using dna probes

genetic screening and genetic fingerprinting both use dna probes
in genetic screeing probes used to locate genes, in genetic fingerprinting porbes used to locate core sequences in introns which are at the same locations in all individuals but the number of times their repeated varies in each individual

below is just a summary of where each of the different features are in different processes

primers-
pcr
sanger method

dna probes
genetic fingerprinting
restriction mapping
locating a gene

pcr
genetic fingerprinting
sanger method
genetic scrreening- te replicate the probe
in vitro cloning- it is invitro clongin

hybridisation
locating genes- dna probes
genetic fingerprintin- dna probes


restriction endonucleases-
restriction mapping
in viv gene cloning
genetic fingerprintin
used in gene therapy to cut out the gene etc and insert it as a vector-



gel electrophoresis-
dna fingerprining
sequencing dna
restrcion mapping
locating a gene uv /xray

Ok, but why isn't it a 3kb fragment, then a four one? I get the impression I'm missing the obvious here. There's four total digest, so three cut sites. One at 3, how do you know where the other two are?

O------3------------4---------------


Sorry for sounding really stupid...

Random stuff I have seen on TV most likely in very what seemed pointless at the time but are now life saving documentories

Probes: first of all, the definition for probes that seems to come across most often is short, single strands of DNA that have a base sequence complementary to the base sequence of part of the target gene. The probe can then bind to (or hybridise with) the target gene. Probes are labelled, usually radioactively (so to see where the probes have attached, it's exposed onto x-ray or photographic film) or with a fluorescent label (so the target gene will fluoresce under UV light). In a practical sense, this is used with restriction endonucleases to break the DNA sample into fragments, and then transferred to a nylon membrane (southern blotting) and incubated with the probe. If the gene is present, it will be labelled and can be detected. (To carry on this idea, if a disease causing gene/gene mutation has been identified, the best course of treatment can be decided.)

On a large scale, probes are used for genetic screening for mutated genes as a part of a DNA microarray, where DNA is washed over an array of different DNA probes (for different mutated genes) and if the DNA binds to a specific probe, the mutation can be identified. [Note: in the specification, you're supposed to understand that scientific methods are constantly updated. This is an example, as DNA microarrays are much faster and cost-effective than using DNA probes on a smaller scale.] This again lets you consider treatments, and you can also bring up genetic counselling (e.g. whether or not to screen embryos if parents are a carrier for a mutated gene).

Restriction mapping, meanwhile, is kind of horrible. First of all, restriction mapping is used for putting DNA fragments back in the right order, because in order for DNA sequencing to take place, the (labelled, this is important) DNA sample must first be cut into fragments using restriction endonucleases, and then separate the fragments by length using electrophoresis.

You then get a nice little (haha, no) diagram:



This is a pretty simplified version; it's possible you can get one with more than one restriction enzyme, but for let's keep it simple for now. Your total digest fragments are ones that have been completely cut by the enzyme (which I'm going to call enzyme A). Your partial digest fragments occur when the enzymes haven't been left long enough, so you can also get fragments of different lengths (in this instance, there isn't a cut after the 3kb fragment, so you have a fragment of 8kb because 3+5=8 - and in the mark schemes, they do apparently like to see you can add 3+5). The radioactive fragment is important, as this gives you your starting point - this is because the radioactive fragment is the one with the probe attached - so in this instance, because we have a partial fragment of 8 and not 7 (and so not 2+5), there are two recognition sequences and so the recognition map is...



(Concise posts - this is how not to do it. )

what time are people sleeping tonight. Or are some staying up late for some last minute cramming session especially for the essay :/

ave any teachers given hints on the essay topic?????????

Thanks but I don't get when you say there isn't a cut after 3kb in the partial digest

on the speciman paper, question 5a i it says explain the shape of the action potential curve. When the action potential increases surely this is due to the the sodium ion channels opening causing sodium ions to diffuse INTO the axon, but the mark scheme says that the sodium ions are diffusing OUT of the axon???

You need to sequence the target gene that you want to make probes for.

Sequencing allows you to work out the base sequence of a piece of DNA via the chain termination method, (CTM) (addition of modified labelled nucleotides).

Synopsis of CTM

Single stranded DNA template, DNA polymerase, DNA primers, free nucleotides and fluorescently labelled modifed nucleotides are added to each of four tubes.

When a modified nucleotide is added to a DNA strand, it halts the addition of any more bases, and so 'terminates' the hybirdisation.

Tubes undergo PCR, produces many strands of different length, (each terminates at different point, depending on where modified nucleotide added).

DNA fragments in each tube undergo electrophoresis are are visualised under UV (modified nucleotdies fluroescently labelled).

Complementary base sequence can be read from the gel, smallest nucleotide (one base) at bottom of gel, each band after this represents on more base added, by reading bands from bottom of the gel to the top, you can determine the DNA base sequence.

Once you have the sequence of your target gene, you can then know the base sequence required for a complementary probe.

So if base sequence is: ATGTTCG

and you want a probe of four nucleotides long, it would be: TACA, as T is complementary to A (first nucelotide in DNA sequence).

Hope that helped

I'm theoretically having an early night, which means I'm going to spend hours rolling around in my bed unable to sleep. Basically accepted I will suck at the essay unless I get super lucky.

hey guys.. you know the sanger method... i dont quite get it..
ive got a question...
dna sequence is tggtcacga and it says give the shortest dna fragment which would be produced when modified cytosine is added. the answer is TC
how do i do this type of question?