In the university genetic engineering exam, there are three big questions for experts to answer! Urg

4.Using mRNA as a template, catalyzed by reverse transcriptase, reverse transcribed into cDNA in vitro, and ligated with an appropriate vector (commonly used phage or plasmid vector) to transform the recipient bacteria, each bacterium contains a piece of cDNA and can multiply and expand, so that the collection of cDNA clones containing all the mRNA information of the cell is called the cDNA library of the tissue cell.

Prokaryotes do not have a polya tail and are not easily reverse transcribed.

Methanol can be used as a carbon source quickly, while MUSES cannot use methanol as a carbon source. Therefore, MUT+ can also grow rapidly in methanol (mm) plates, while MUTS can only grow rapidly in glucose (MD) plates.

6.Nick translation is a method that uses the enzymatic activities of Escherichia coli DNA polymerase I to incorporate labeled DNTPs into newly formed DNA strands to form uniformly labeled high-specific active DNA probes. Here's how it works:

1) Dissolve 1ug of DNA in a small amount of sterile double distilled water and add 5?Approximately 10 incision translation buffer (Tris HCl,; mol/l mgso4 ；1 mmol l dithiothreitol; 500?g ml bovine serum albumin), add the marker (eg?).

32 p-datp) and 1 μl solution of the other three dntps (e.g., DCTP, DGTP, DTTP) and 20 mM L solutionl。

2) Add 100?ci(10?l) marker solution, add sterile double distilled water to the final volume, mix well, add diluted dnasei solution, mix well; Add 1 ul (about 5 units) of DNA polymerase I and mix well.

3) Allow for 14-16 hours of reaction.

4. Look at high school biology elective three.

1) In genetic engineering.

There are two main ways to obtain the target gene, namely artificial synthesis and isolation from biological materials, artificial synthesis can be achieved by reverse transcription or artificial chemical synthesis, and isolation from biological materials is through gene library method

2) Since the cDNA library only contains the genes that have been transcribed (or expressed) by leaf cells, while the genomic library contains all the genes of the plant, the number of genes contained in the two libraries is less than that in the genomic library compared with the basal surplus

3) The composition of the gene expression vector includes the target gene + promoter.

Terminator + marker gene, where the promoter is RNA polymerase.

the site of identification and bonding; In the case of prokaryotes.

Receptor cells, which are gene expression vectors, are most commonly used in prokaryotes such as Escherichia coli due to the rapid reproduction rate of bacteria

4) Genetic guns are monocots.

A gene transformation method commonly used in China, the commonly used metal particles carrying the target gene are gold dust and tungsten powder particles, and the cost is high

So the answer is: 1) Synthetic biomaterials.

2) The cDNA library contains only the genes that have been transcribed (or expressed) by leaf cells, while the genome library contains all the genes of Zheng Chan for the plant.

3) RNA Escherichia coli (or A. bacteria).

4) Gold powder: tungsten powder.

Core step: Construction of gene expression vectors.

Principles of genetic engineering: genetic recombination.

Reasons for success: The DNA of different organisms has the same material basis (all with deoxynucleotides as the basic unit) and structural basis (all have the spatial structure of the double helix), and almost all organisms share a set of genetic code.

The principle of PCR: DNA replication.

What is the basis for the primer to bind to the PCR template during renaturation? What do you want to ask this question that I don't understand, the structure is hydrogen bonding, and the principle is the principle of base complementarity, and the method is to cool down to 72-75

The most important feature of DNA polymerases used in routine PCR amplification is that they are resistant to high temperatures.

The integration site of the gene of interest is random, and it is not necessary for only a single plasmid to be integrated, but it is also possible that multiple loci are integrated, such as the integration of 2 resistance genes, one may be on a pair of homologous chromosomes, and the other may be on two non-homologous chromosomes. In the case of the former, the plant will become homozygous (RR) for resistance, and the offspring should be all genotype and phenotype resistant relative to the wild-type plant (RR). If the latter, although the transformed plant has 2 resistance genes but is located on the non-homologous chromosome, it is equivalent to the free combination between the non-homologous chromosome genes during crossing, as long as the gamete contains the resistance gene (R) and combines with the wild-type gamete (R), the resistance plant will be produced, the probability of 2 R to a gamete is 25%, the probability of one R is 50%, and the probability of 1 R is 25%, so the post-representative type will have a 3:1 segregation ratio, This is the same as the result of crossing Plant 2 with the wild type.

Plant 1 should have integrated only one R into the genome. This method of hybridization with recessive homozygotes is assay crossing. Resistant plant selection is to inoculate explants into a medium with kanamycin for screening.

This pair of relative traits conforms to Mendel's law of separation and the law of free combination. The wrong one is b.

Analyzing the problem, from the ratio of the number of offspring resistant to non-resistant is 1:1 and 3:1 respectively, and because the wild-type (non-resistant) AA, it can be inferred that the resistance is dominant, and A is correct.

b If they are all on homologous chromosomes, then the genotype of plant 2 is AA, and the offspring crossed with AA are all disease-resistant, and B is wrong. c. Cells that have obtained disease resistance genes should be cultivated by plant tissue culture d. Genetic engineering is that DNA is heritable, so Mendelian laws of inheritance are followed. If there's anything wrong, tell me. Thank you.

b If it is on the homologous chromosome, it will not be 3:1 in this case, only AA*AA will do, but the wild type is homozygous ......

Your doubts are right and understandable. Personally, I think that the author of the question means that there are these two enzyme sites at both ends of the target gene, and these enzyme sites can be separated. For example--- e1-e1, e1--b1---e1, e1-b1---e1-b1, b1--e1---b1-e1

Ideally, double digestion yields only one gene. However, if the digestion sites overlap or are in close proximity, the digestion will not be complete as you fear, resulting in complex multiple products.

Personally, I think that the author of the question means that there are these two enzyme cleavage sites at both ends of the target gene, and these cleavage sites can be completely separated. But the person who wrote the question did not have you to think about it comprehensively, or lacked practical work experience. Because in the actual work, the problems you worry about often arise and affect the work efficiency.

You are better than the person who wrote the question. Blue is better than blue!

Multiple choice questions: The bacterial plasmid molecules commonly used by people to carry genetically engineered delivery vehicles often carry antibiotic resistance genes,1Some genes of interest are not directly expressed in the trait of the organism, so the purpose may not be visible.

Restriction enzymes are used for plasmids and restriction enzymes are used for target genes. Because the gene of interest needs to be cut on both sides, the answer is obviously to use restriction enzymes. Then there are two marker genes on the plasmid, and if the restriction enzyme is also used, then both marker genes will be destroyed and will not be useful in the next step of screening.

So use restriction enzyme 1 to cut. Then, the recombinant plasmid that can survive in ampicillin-containing medium, but not tetracycline-containing medium, is needed.

It's simple, if I go back to high school, but now I'm graduating from college early... Forgot about the dear.

a.A marker gene is a gene with a known function or a known sequence that can play a specific role as a marker. In the sense of genetic engineering, it is an important marker for recombinant DNA vectors, which is usually used to test the success of transformation. In the sense of gene mapping, it is a tool for marking the gene of interest, which is usually used to detect the localization of the gene of interest in cells.

So a mistake. b.Reverse transcription is to use the messenger RNA transcribed from the target gene as a template, reverse transcribed into complementary single-stranded DNA (cDNA), and then synthesize double-stranded DNA under the action of enzymes to obtain the desired gene.

It is one of the important methods for extracting the target gene in genetic engineering.

Insulin can be extracted from pancreatic B cells, so messenger RNA can be extracted as a template.

So B right. c.Introducing the gene of interest into the recipient cell is the third step in implementing genetic engineering.

So C is wrong. d.Genetic engineering involves a wide range of tool enzymes, which can be roughly divided into four categories: restriction enzymes, ligases, polymerases, and modified enzymes. Among them, restriction endonucleases and DNA ligases play the most prominent role in molecular cloning.

So d wrong. I hope to answer your doubts about biology and hope to help you get good results in this subject!

If the target gene is selected incorrectly, it is only convenient to screen the target gene, but cannot promote the expression of the target gene.

B is correct, pancreatic B cells secrete insulin, so the cells contain messenger RNA that synthesizes insulin;

c Error, not a genetic mutation, but a genetic recombination;

D is false, not DNA polymerase, is DNA ligase.

Hope it helps. ^_