Hot Crick Strand Dna Case Study
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SN1 mapping allows you to see where transciption starts He wanted to show that txn starts a set distance from the TATA box in a class 2 promoter. He got a piece of DNA that had a good class 2 promoter and a piece of DNA that he knew he could cut with restriction endonucleases. The idea is to pair a piece of DNA with label on only one strand. In this case you want the label on the crick strand. You would take this piece of DNA with a hot phosphate on 5 prime end of the crick strand and denature it. The probe DNA helps you see where txn starts. You take that probe DNA and you mix it with transcript thats made by the enzyme. He lets the enzyme make cold mRNA and then mixes the cold mRNA with the probe and then adds S1 nuclease. S1 nuclease will cut anything that is single stranded. Part of the probe is single stranded, so your probe will get smaller. How small your probe will get depends on where transcription starts. This was the procudure that Chambone used to show that RNA polymerase 2 keys on the position of the TATA box. RNA pol …show more content…
Whats important is that you know the sequence of the DNA and you have made yourself a piece of HOT crick strand DNA. In SN1 mapping you call you hot crick strand is called a probe. In Primer Extension, your hot crick is not a probe, its a primer. Its a primer for reverse transcriptase. Which is an enzyme that can read RNA and make DNA. Which is way primer extension is possible, because of reverse transcriptases that come from tumor causing retroviruses. Whats special about the enzyme is its ability to read a mRNA, and extend a primer strand (in this case its a hot crick DNA). Reverse transcriptase can find that 3 prime hydroxyl and use the mRNA as a template. The hot crick DNA will be extended out to the point where reverse transcritase reaches the end of the message. The size depends on where the message starts because it will read the message all the way to the 5 prime terminal end of the
SN1 mapping allows you to see where transciption starts He wanted to show that txn starts a set distance from the TATA box in a class 2 promoter. He got a piece of DNA that had a good class 2 promoter and a piece of DNA that he knew he could cut with restriction endonucleases. The idea is to pair a piece of DNA with label on only one strand. In this case you want the label on the crick strand. You would take this piece of DNA with a hot phosphate on 5 prime end of the crick strand and denature it. The probe DNA helps you see where txn starts. You take that probe DNA and you mix it with transcript thats made by the enzyme. He lets the enzyme make cold mRNA and then mixes the cold mRNA with the probe and then adds S1 nuclease. S1 nuclease will cut anything that is single stranded. Part of the probe is single stranded, so your probe will get smaller. How small your probe will get depends on where transcription starts. This was the procudure that Chambone used to show that RNA polymerase 2 keys on the position of the TATA box. RNA pol …show more content…
Whats important is that you know the sequence of the DNA and you have made yourself a piece of HOT crick strand DNA. In SN1 mapping you call you hot crick strand is called a probe. In Primer Extension, your hot crick is not a probe, its a primer. Its a primer for reverse transcriptase. Which is an enzyme that can read RNA and make DNA. Which is way primer extension is possible, because of reverse transcriptases that come from tumor causing retroviruses. Whats special about the enzyme is its ability to read a mRNA, and extend a primer strand (in this case its a hot crick DNA). Reverse transcriptase can find that 3 prime hydroxyl and use the mRNA as a template. The hot crick DNA will be extended out to the point where reverse transcritase reaches the end of the message. The size depends on where the message starts because it will read the message all the way to the 5 prime terminal end of the