What Efficiency Is Needed in DNA Synthesis for High Yield?

[Strife]

I am new to this site, so if I make a mistake with the format, I apologize.

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**Question #1**:

Homework Statement

1. You are trying to make a 100-nucleotide long chain of DNA using a DNA synthesizer machine. What must be the minimum efficiency of each coupling (nucleotide addition) step, so that the full-length product chain is at least 25% of all the DNA chains synthesized (i.e., failure sequences must not be >75% of the total)?2. Homework Equations and the attempt at a solution

I thoroughly understand the mechanism by which the DNA synthesizer machine works, but am not sure how to calculate efficiency. No further information was given in the question, and the only information I could find on google/in my notes was just explaining how the machine works. Any help/thoughts would be greatly appreciated.------------------------------------------------------------------------------------------------------------

**Question #2**:

Homework Statement

How would you replace a mouse gene by the wild type copy of a human gene?2. Homework Equations and the attempt at a solution

I know that the answer is using the Cre-loxP system. I don't, however, know the exact steps to doing a translocation using this method. I found multiple sites that give the steps for a deletion, but none that went into any detail about a translocation.---------------------------------------------------------------------------------------------------------------

**Question #3**:

Homework Statement

A cDNA library is made with mRNA isolated from liver tissue. When a cloned cDNA from the library is digested with restriction enzymes EcoRI (E), HindIII (H), and BamHI (B), the restriction map shown in Fig. (a) is obtained. When this cDNA is used to screen a cDNA library made with mRNA from brain tissue, 3 identical cDNAs with the restriction map shown in Fig. (b) are obtained. When either cDNA is used to synthesize a uniformly labeled radioactive probe and the probe is allowed to hybridize to a Southern blot prepared from genomic DNA digested singly with the enzymes EcoRI, HindIII or BamHI, an autoradiogram shown in Fig. (c) is obtained. When either of these cDNA-derived radioactive probes is used to hybridize to a Northern blot prepared with poly (A)+ RNA isolated from liver and brain tissues, the pattern of bands in Fig. (d) is seen. Fully analyze these data and then answer the questions below

a. Do the cDNAs derive from the same gene?
b. Why do the cDNAs have different restriction maps?
c. Why are different sized bands seen in the Northern blot?
d. Why do some of the bands seen on the whole genome Southern blot have different sizes than some of the restriction fragments in the cDNAs?

The figures for this question can be found at: http://photos-g.ak.facebook.com/photos-ak-sf2p/v360/61/45/8826787/n8826787_42325350_4285.jpg
2. Homework Equations and the attempt at a solution

I have a guess for each of the four answers, but ultimately that's all it is: a guess. If anyone would be willing to either confirm/correct any of the guesses, that'd be greatly appreciated.

a. yes
b. alternative splicing
c. due to an intron
d. the restriction map may not contain the whole gene

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Thank you!

 

[nate808]

Hello,

Welcome to the forum and thank you for your questions. I will do my best to help you understand and find solutions to your problems.

For Question #1, the minimum efficiency of each coupling step can be calculated using the formula: Efficiency = (successful couplings)/(total couplings) x 100%. In this case, the efficiency should be at least 25% to ensure that the full-length product chain is at least 25% of all the DNA chains synthesized. This means that at least 25 out of 100 nucleotides must be successfully added in each step. If we assume that each coupling step has the same efficiency, then the minimum efficiency for each coupling step would be 25%.

For Question #2, you are correct in saying that the Cre-loxP system can be used to replace a mouse gene with the wild type copy of a human gene. The steps for this are as follows:

1. Design a targeting vector that contains the human gene flanked by loxP sites.

2. Inject the targeting vector into mouse embryonic stem cells.

3. Use Cre recombinase to remove the mouse gene and replace it with the human gene.

4. Select for cells that have successfully incorporated the human gene.

5. Inject these cells into mouse embryos and transfer the embryos into a surrogate mother.

6. The resulting mice will have the human gene instead of the mouse gene.

For Question #3, here are the answers to the four parts:

a. Yes, the cDNAs are likely from the same gene because they have the same restriction map.

b. The cDNAs have different restriction maps due to alternative splicing. This means that different exons may be included or excluded in the cDNA sequences, resulting in different restriction maps.

c. The different sized bands seen in the Northern blot are likely due to alternative splicing as well. This means that the mRNA transcripts from the same gene may have different sizes due to the inclusion or exclusion of different exons.

d. The bands seen on the whole genome Southern blot may have different sizes than some of the restriction fragments in the cDNAs because the restriction map may not contain the entire gene. This means that there may be other regions of the gene that are not included in the restriction map and therefore not detected on the Southern blot.

I hope this helps. If you have any further questions or need clarification, please don't hesitate to ask. Good luck

 

[Blueshift5]

I appreciate your questions and your attempt at solving them. Here are my responses to each of your questions:

Question #1:
Efficiency in this case refers to the percentage of successful couplings (nucleotide additions) compared to the total number of attempted couplings. To calculate this, you would need to know the total number of nucleotides added and the total number of chains synthesized. From there, you can calculate the efficiency using the formula: (successful couplings/total number of attempted couplings) x 100. In order to achieve a full-length product chain that is at least 25% of all the DNA chains synthesized, the minimum efficiency for each coupling step would need to be at least 75%.

Question #2:
To replace a mouse gene with a wild type copy of a human gene, you would need to use the Cre-loxP system. This involves using a Cre recombinase enzyme to excise the mouse gene and replace it with the wild type human gene. The steps involved would include designing a targeting vector containing the human gene flanked by loxP sites, introducing this vector into mouse embryonic stem cells, and then using Cre recombinase to excise the mouse gene and insert the human gene. This process is known as gene targeting and is commonly used in genetic engineering.

Question #3:
a. Based on the information provided, it is likely that the cDNAs do derive from the same gene. This is because they have the same restriction map when digested with the enzymes EcoRI, HindIII, and BamHI.

b. The cDNAs have different restriction maps because they are derived from different mRNA transcripts. This can be due to alternative splicing, where different exons are included or excluded from the final mRNA transcript.

c. The different sized bands seen in the Northern blot are likely due to alternative splicing, where different exons are included or excluded from the final mRNA transcript.

d. The bands seen on the whole genome Southern blot may have different sizes compared to some of the restriction fragments in the cDNAs because the restriction map may not contain the entire gene. This could also be due to mutations or polymorphisms in the gene that result in different restriction sites.