BIOL 3110 Lecture Notes - Okazaki Fragments, Polynucleotide, Reverse Transcriptase

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maroonskunk776

7 Oct 2012

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Used in analyzing a digest fragment with a p32 probe, used for dna fragments. Mammalian genomes are relatively large, with ~ 3 x 109 bp, the frequency of finding a desired site is dependent on how many bases the restriction enzyme requires (i. e. ecor1 recognizes 6bps, therefore, (1/4)6 = 1/4096) Once the fragments are processed in the gel it needs to be transferred onto a nitrocellulose filter and needs to be exposed via radiography. Similar to the southern blot but this analyzes rna. 5" -----------------aaaaaa 3" (3"oh)t t t t t t annealing olgio primer reverse transcriptase + 4 dntp"s. Rnase h (digests rna when rna/dna or alkaline) (3"oh) Dna polymerase + 4 dntp + reverse transcriptase. S1 nuclease (digests ss dna or ss rna cdna probe with frayed ends. There are a few problems with these types of synthetic dna: Both ends of the dna probe are frayed. S1 nucleases will start to eat away the open ends.

Related Questions

1. A study was conduced to identify a liver cell gene (zed) that has a length of 3000 bp. A southern blot was prepared using DNA from the liver cell and a probe made to region 1600-2000 of zed. Analysis of the blot revealed that there are two spots present, one at 3000 and another at 2000. A plausible explanation for this observation is

A. there is an identical copy of this gene on another chromosome.

B. the gene was cut into two equal portions by a restriction enzyme.

C. there is another gene with sequence similar to gene zed

D. both: there is an identical copy of this gene on another chromosome and there is another gene with sequence similar to gene zed

E. no explanation can be given using the available data

2. Protein P is synthesized in relatively high amounts in human pancreas. This protein has been isolated and purified, but its amino acid sequence has not been determined. We wish to clone the gene for protein P.

(a) How can a probe be prepared to identify the gene for protein P?

(b) If we have prepared a radioactive messenger RNA as our probe in part(a), how could we verify that it is the mRNA for protein P?

(c) If we wish to make a gene library from the DNA of human pancreatic cells, we proceed by digesting the DNA with the restriction enzyme BamHI and then separating the fragments by agarose gel electrophoresis. The fragments are transfered (by southern blotting) onto a nitrocellulose filter and flooded with the radioactive probe. Following incubation, the filter is washed to remove any probe that has not specifically bound to one or more DNA fragments. Autoradiography of the filter reveals two bands of size 300 and 700 bp. When experiment is repeated using restraction enzyme PstI, only one band of 1000 bp appears. If it is already known that pancreatic cells contain a single copy of the gene of protein P, which of these enzymes should be used to construct our gene library?

violethornet799

DNA Fingerprinting III: Southern Blotting Questions

A) Why do different individuals such as siblings have differentrestriction enzyme recognition sites?

B) What is the function of probes in DNA praternityanalysis?

C) Why is there more than one single locus probe used in anactual paternity DNA test?

D)What advantage does Southern Blot Analysis affer for genetictesting?

Thank you very much!

pinkduck621

1. What information would you get from a northern blot that youwould not get from reverse transcriptase-PCR if used to detect thesame mRNA?

2. What information would you get from a Southern blot that youwould not get from PCR experiment if used to detect the samegenetic locus in human genomic DNA?

3. Given your answers to A and B, why is it more common tochoose to use the PCR based methods instead of the blot basedmethods?

4. What information would you get from quantitative (real-time)reverse transcriptase-PCR that you would not get from conventionalreverse transcriptase-PCR when used to analyze the same mRNA?

5. What information would you get from in situ hybridizationthat you would not get from a northern blot when used to analyzethe same mRNA?

6.What information would you get from a western blot that youwould not get from a northern blot when used to analyze theexpression the same gene?

rubyturtle713

BIOL 3110 Lecture Notes - Okazaki Fragments, Polynucleotide, Reverse Transcriptase

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