Recombinant DNA technology - 4 page summary - includes out of spec content - PCR -reverse transcriptase - fingerprinting and more

What is recombinant DNA and why does it work?

Respuesta: Recombinant DNA technology involves the transfer of fragments of DNA from one organism to another, so recombinant DNA is combination of the DNA of two different organisms. The genetic code is universal, so despite the DNA coming from another organism, the transferred DNA can be transcribed and translated within the transgenic organism.

How to isolate a gene from e.g. human tissue? (name 3 methods)

Respuesta: Reverse transcriptase, gene machine, and restriction endonucleases

Describe conversion of mRNA to cDNA using reverse transcriptase.

Respuesta: First, remove the mRNA that codes for the specific polypeptide from the host cell. The mRNA strand acts as a template, on which complementary DNA nucleotides attach, and reverse transcriptase is used to produce a single strand of cDNA (complementary DNA). The mRNA strand is hydrolysed off using an enzyme. DNA polymerase builds up complementary DNA nucleotides on the cDNA template and eventually forms a double stranded DNA sequence of the required gene.

How to manufacture a gene e.g. using the gene machine?

Respuesta: The desired base sequence is determined by using the triplet codes of the amino acids used to make the polypeptide. The desired base sequence is fed into a computer, and it designs a series of small, overlapping, single-strands of nucleotides, called oligonucleotides. The oligonucleotides are the assembled, adding one nucleotide at a time to make the sequence. These are then joined together to make a gene with no introns. It is made double stranded using the polymerase chain reaction, which constructs a complementary strand. It is then replicated, again using the polymerase chain reaction. Advantages: Sequence of nucleotides can be produced quickly (in 10 days), and there are no introns present, so can be translated by prokaryotes.

How do you isolate a gene using restriction enzymes and insert the desired gene into the plasmid?

Respuesta: Restriction endonucleases cut the DNA in a staggered fashion at a specific sequence of bases (recognition sequence) to form sticky ends. The unpaired bases at the cut end are palindromes of each other. Afterwards, a promoter region is added to the start of the DNA fragment so transcription factors and RNA polymerase can attach to initiate transcription, and a terminator region is added to the end of the DNA fragment to stop transcription. A vector is the carrying unit for the desired gene. Usually, it is a plasmid from a bacterium that codes for an antibiotic resistance gene. The same restriction enzyme is used to break up the antibiotic resistance gene in the plasmid loop, so the sticky ends are complementary to the cut out desired gene. The DNA fragments are mixed with the open plasmids in hopes some may become incorporated in them. The complementary bases in the sticky ends pair up and DNA ligase forms the sugar-phosphate backbone between the two sections of DNA. This forms recombinant DNA in the plasmid. Using the same restriction endonuclease is important to make complementary sticky ends, so that the DNA of one organism can combine with the DNA of another.

What are the outcomes of trying to insert the desired gene into the plasmid?

Respuesta: Not all plasmids take up the desired gene, because some plasmids close up again without incorporating the gene. Some of the desired genes close up in a small circle, creating their own mini plasmid. Finally, the remaining few plasmids do successfully take up the desired gene.

What is Transformation?

Respuesta: Transformation is the process of incorporating some of the plasmids with the recombinant DNA into the bacterial cells. There are several methods to achieve this: The plasmids and bacterial cells are mixed together in a medium containing calcium ions, which make the cell membrane of the bacterium more permeable to allow them to take up the plasmids. Heat shock, so the bacteria is subject to fluctuating temperatures between 0°C and 42°C so the cell walls and cell membranes become more permeable, which allows the plasmid to enter more easily. Electroporation, where a high voltage is applied to the bacterial cell, making the cell membrane more permeable so the plasmid can be taken up more easily.

MARKER GENES: How do you identify which bacterial cells have taken up the plasmid which contains the desired gene?

Respuesta: The bacteria that have taken up the desired gene will no longer be resistant to the antibiotic that is coded for by the plasmid loop, as it has been broken. Often the plasmid used has 2 antibiotic resistance genes, one that is always present in the plasmid, and the other that gets disrupted if it takes up the desired gene. Hence, the bacteria that take up any sort of plasmid will always be resistant to the first antibiotic, and the bacteria that don’t take up any plasmids or just a loop of the desired gene are killed when using this antibiotic. Afterwards, the bacteria that take up the plasmid that have the desired gene incorporated in them, they are not resistant to the second antibiotic. Using replica plating, the bacteria that are only resistant to the first gene can be identified, and they are the bacteria containing plasmids which contain the desired gene. Alternatively, the desired gene could also be attached to an identification gene, such as a fluorescent marker, so the bacteria that take up the desired gene fluoresce under a microscope making it easier to distinguish. Or the desired gene could be attached to the gene which produces lactase. Lactase turns a particular colourless substrate blue, so the bacteria which take up the desired gene are able to change the substrates colour.

How does the Polymerase Chain Reaction (PCR) work?

Respuesta: PCR is a method of copying fragments of DNA. 4 things need to be entered into the machine (thermocycler): the DNA fragment to be copied; DNA polymerase, more specifically taq polymerase obtained from bacteria in hot springs as they are at their optimum at high temperatures of 72°C to join nucleotides together by forming phosphodiester bonds; 2 primers, short sequences of nucleotides that have a set of bases complementary to those at the start of each of the two DNA fragment strands, one acting 3’ to 5’, and the other 5’ to 3’; and many nucleotides, A,T,G,C. After these are entered, the temperature is increased to 95°C which breaks the hydrogen bonds between the DNA fragments, separating them into two strands. The mixture is then cooled to 55°C so the primers can attach to the DNA strands (so they don’t simply rejoin), and so they provide a starting region for the DNA polymerase to bind. The temperature is then increased to 72°C so the DNA polymerase can add complementary nucleotides along the separated strands until it reaches the end of the chain. The cycle can then be repeated, multiple times.

Why is PCR used?

Respuesta: To amplify a DNA sample, e.g. one found at the crime scene so further tests can be conducted on it. Advantages and disadvantages of in vitro cloning VS advantages and disadvantages of in vivo cloning: PCR is automated, making it rapid and efficient. It is particularly valuable for forensics. It also does not require living cells, so no complex culturing techniques that require time and effort are needed. PCR requires a very pure sample, any contamination is also amplified and can lead to false results from further testing etc. It is also less accurate, and any errors in copying DNA are also copied in the subsequent cycles. In vivo cloning is particularly useful when the main objective is to introduce the desired gene into an organism, e.g. introducing the vector to a human. Additionally, it involves almost no risk of contamination as the desired gene has been cut using the same sticky ends so contaminant DNA will not be taken up by the plasmid. Very accurate, the chances of a mutation are very rare. It also cuts out a specific gene, so not the whole DNA sample is being replicated. It also produces transformed bacteria, which is useful if the main objective is to produce proteins commercially/ medically e.g. insulin. In vivo cloning is very time consuming. It could take many days or weeks to produce the same quantity of DNA that PCR would in a shorter time.