Explanation of basic molecular biology terms
1. cDNA and cccDNA: cDNA is a double-stranded DNA synthesized by reverse transcriptase from mRNA; cccDNA is a plasmid double-stranded closed circular DNA free from the chromosome.
2. Standard folding unit: the protein secondary structure unit α-helix and β-sheet can form structural blocks with special geometric arrangements through various connecting polypeptides. This type of determined folding is usually called super secondary structure. Almost all tertiary structures can be described by these folding types, and even their combined types, so they are also called standard folding units.
3. CAP: cyclic adenosine monophosphate (cAMP) receptor protein CRP (cAMP receptor protein), the complex formed after the combination of cAMP and CRP is called activating protein CAP (cAMP activated protein)
4. Palindromic sequence: The reverse complementary sequence of a segment of a DNA fragment, often a restriction enzyme site.
5. micRNA: Complementary interfering RNA or antisense RNA, which is complementary to the mRNA sequence and can inhibit the translation of mRNA.
6. Ribozyme: RNA with catalytic activity, which plays an autocatalytic role in the splicing process of RNA.
7. Motif: There are some local regions with similar three-dimensional shape and topology in the spatial structure of protein molecules
8. Signal peptide: a peptide with 15-36 amino acid residues at the N-terminus during protein synthesis, which guides the transmembrane of the protein.
9. Attenuator: A nucleotide sequence between an operator region and a structural gene that terminates transcription.
10. Magic Spot: When the bacteria grows and encounters a complete lack of amino acids, the bacteria will produce an emergency response to stop the expression of all genes. The signals that generate this emergency response are guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp). The role of PpGpp and pppGpp is not just one or a few operons, but affects a large number of them, so they are called super-regulators or magic spots.
11. Upstream promoter element: refers to the DNA sequence that plays a regulatory role in the activity of the promoter, such as TATA in the -10 region, TGACA in the -35 region, enhancers, and attenuators.
12. DNA probe: a labeled segment of DNA with a known sequence, which is widely used to detect unknown sequences and screen target genes.
13. SD sequence: It is the binding sequence of ribosome and mRNA, which regulates translation.
14. Monoclonal Antibody: An antibody that acts only against a single antigenic determinant.
15. Cosmid: It is an artificially constructed exogenous DNA vector that retains the COS regions at both ends of the phage and is connected to the plasmid.
16. Blue-white spot screening: LacZ gene (encoding β-galactosidase), the enzyme can decompose the chromogenic substrate X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) to produce blue, thus making the strain blue. When the exogenous DNA is inserted, the LacZ gene cannot be expressed, and the strain is white, so as to screen the recombinant bacteria. This is called blue-white screening.
17. Cis-acting element: A specific sequence of bases in DNA that regulates gene expression.
18. Klenow enzyme: Large fragment of DNA polymerase I, except that the 5' 3' exonuclease activity is removed from the DNA polymerase I holoenzyme
19. Anchored PCR: used to amplify the DNA of interest with a known sequence at one end. A poly-dG tail was added to one end of the unknown sequence, and then the poly-dC and the known sequence were used as primers for PCR amplification.
20. Fusion protein: The gene of eukaryotic protein is connected with exogenous gene, and the protein composed of the translation of original gene protein and exogenous protein is expressed at the same time.
Other molecular biology terms
1. The physical map of DNA is the order in which the (restriction endonuclease-digested) fragments of the DNA molecule are arranged.
2. The cleavage of RNase is divided into two types (autocatalysis) and (heterocatalysis).
3. There are three initiation factors in prokaryotes are (IF-1), (IF-2) and (IF-3).
4. Transmembrane proteins require guidance (signal peptides), and the role of protein chaperones is (helps to fold the peptide chain into the native conformation of the protein).
5. Elements in promoters can generally be divided into two types: (core promoter elements) and (upstream promoter elements).
6. The research content of molecular biology mainly includes three parts: (structural molecular biology), (gene expression and regulation), and (DNA recombination technology).
7. The two key experiments demonstrating that DNA is the genetic material are (pneumococcus infection of mice) and (T2 phage infection of Escherichia coli). potential).
8. There are two main differences between hnRNA and mRNA: (hnRNA is spliced in the process of converting into mRNA), (the 5' end of the mRNA is added with an m7pGppp cap, and there is an extra polyadenylation at the 3' end of the mRNA acid (polyA) tail).
9. The advantages of the multi-subunit form of protein are (subunit is an economical method for DNA utilization), (can reduce the impact of random errors in protein synthesis on protein activity), (activity can be very efficiently and rapidly are opened and closed).
10. The main content of protein folding mechanism first nucleation theory includes (nucleation), (structural enrichment), (final rearrangement).
11. Galactose has a dual effect on bacteria; on the one hand (it can be used as a carbon source for cell growth); on the other hand (it is also a component of the cell wall). Therefore, a cAMP-CRP-independent promoter S2 is needed for the permanent synthesis at the background level; at the same time, a cAMP-CRP-dependent promoter S1 is needed to regulate the high-level synthesis. Transcription starts from ( S2 ) with G and from ( S1 ) without G.
12. Recombinant DNA technology is also known as (gene cloning) or (molecular cloning). The ultimate goal is (to transfer the genetic information DNA in one organism into another organism). A typical DNA recombination experiment usually includes the following steps: (1) Extract the target gene (or exogenous gene) of the donor organism, and enzymatically connect it to another DNA molecule (cloning vector) to form a new recombinant DNA molecule. ② The recombinant DNA molecule is transferred into the recipient cell and replicated in the recipient cell. This process is called transformation. ③ Screen and identify those recipient cells that have absorbed the recombinant DNA. ④Cultivate the cells containing recombinant DNA in large quantities to detect whether the foreign aid gene is expressed.
13. There are two types of plasmid replication: those that are strictly controlled by host cell protein synthesis are called (tight plasmids), and those that are not strictly controlled by host cell protein synthesis are called (relaxed plasmids).
14. The PCR reaction system should have the following conditions: a. DNA primers (about 20 bases) with complementary sequences at each end of the two strands of the target gene to be separated. b. Enzymes with thermal stability such as: TagDNA polymerase. c, dNTPd, DNA sequence of interest as template
15. The basic reaction process of PCR includes three stages: (denaturation), (annealing), and (extension).
16. The basic process of transgenic animals usually includes: ①Introduction of cloned foreign gene into the nucleus of a fertilized egg or embryonic stem cell; ②Transplantation of the inoculated fertilized egg or embryonic stem cell into the female uterus; ③Complete embryonic development and growth For the offspring with foreign genes; ④ Use these animals that can produce foreign proteins as breeding stock to breed new homozygous lines.
17. Hybridoma cell lines are generated by hybridizing (spleen B) cells with (myeloma) cells, and since (spleen cells) can utilize hypoxanthine and (bone cells) provide cell division functions, they can be grown in HAT medium. grow.
18. With the deepening of research, the first generation of antibodies is called (polyclonal antibodies), the second generation (monoclonal antibodies), and the third generation (genetic engineering antibodies).
19. At present, the genetic engineering of insect viruses is mainly focused on baculovirus, which is manifested in the introduction of (exogenous toxin gene); (genes that disrupt the normal life cycle of insects); (modification of virus genes).
20. The trans-acting protein factors corresponding to the common elements TATA, GC, and CAAT in the mammalian RNA polymerase II promoter are (TFIID), (SP-1) and (CTF/NF1), respectively.
twenty one. The basic transcription factors of RNA polymerase Ⅱ are, TFⅡ-A, TFⅡ-B, TFII-D, TFⅡ-E, and their binding sequence is: (D, A, B, E). Wherein the function of TFII-D is (binding to TATA box).
twenty two. Most of the transcription factors that bind to DNA work in the form of dimers. The functional domains of transcription factors that bind to DNA are commonly the following (helix-turn-helix), (zinc finger motif), (basic-leucine) zipper motif).
twenty three. There are three types of restriction endonuclease cleavage modes: (cut at the 5' side of the symmetry axis to generate 5' sticky ends), (cut at the 3' side of the symmetry axis to generate 3' sticky ends (cut at the symmetry axis to generate flat segments) ).
twenty four. Plasmid DNA has three different configurations: (SC configuration), (oc configuration), (L configuration). The first in electrophoresis is (SC configuration).
25. Exogenous gene expression systems, mainly (Escherichia coli), (Yeast), (Insect) and (Mammalian cell table).
26. The commonly used methods for transgenic animals are: (retroviral infection method), (DNA microinjection method), (embryonic stem cell method).
Application Molecular biology
1. Name the functions of more than 5 RNAs?
Transfer RNA tRNA Transfer amino acid Ribosome RNA rRNA Ribosome constitutes messenger RNA mRNA Protein synthesis template Heterogeneous nuclear RNA hnRNA Precursor of mature mRNA small nuclear RNA snRNA Involved in hnRNA splicing Small cytoplasmic RNA scRNA/7SL-RNA protein Plasma reticulum-localized and synthesized signal recognition body components Antisense RNA anRNA/micRNA Regulates gene expression Ribozyme RNA Enzymatically active RNA
2. What is the main difference between prokaryotic and eukaryotic promoters?
Prokaryotic TTGACA --- TATAAT------Initiation Site-35 -10 Eukaryotic Enhancer---GC ---CAAT----TATAA-5mGpp-Initiation Site-110 -70 -25
3. What are the main aspects of artificial construction of natural plasmids?
Natural plasmids often have defects, so they are not suitable for use as carriers for genetic engineering, and must be modified and constructed: a. Add suitable selection marker genes, such as two or more, which are easy to use for selection, usually antibiotic genes. b. Increase or decrease suitable enzyme cutting sites to facilitate recombination. c. Shorten the length, cut off unnecessary fragments, improve the import efficiency and increase the loading capacity. d. Change the replicon, from tight to loose, from fewer copies to more copies. e. Add special genetic elements according to the special requirements of genetic engineering
4. Give an example of a method for differential screening of tissue-specific cDNA?
Two cell populations are prepared, the target gene is expressed or highly expressed in one of the cells, and the target gene is not expressed or lowly expressed in the other cell, and then the target gene is found by hybridization and comparison. For example, during the occurrence and development of tumors, tumor cells will present mRNAs with different expression levels than normal cells. Therefore, tumor-related genes can be screened out by differential hybridization. The induction method can also be used to screen out the genes whose expression is induced.
5. Generation and screening of hybridoma cell lines?
Spleen B cells + myeloma cells, add polyethylene glycol (PEG) to promote cell fusion, and the splenic B-myeloma fusion cells grown in HAT medium (containing hypoxanthine, aminopterin, T) continue to expand nourish. The cell fusion contains: spleen-spleen fusion cells: unable to grow, spleen cells cannot be cultured in vitro. Bone-bone fusion cells: cannot utilize hypoxanthine, but can synthesize purine through the second pathway using folate reductase. Aminopterin inhibits folate reductase and thus cannot grow. Bone-spleen fusion cells: can grow in HAT, spleen cells can utilize hypoxanthine, and bone cells provide cell division function.
6. What is the principle and method of determining the primary structure of DNA by the dideoxy terminal termination method (Sanger method)?
The principle is to use a nucleotide chain terminator—2,,3,-dideoxynucleotide to terminate the extension of DNA. Since it lacks the 3-OH required for the formation of 3/5/phosphodiester bonds, once incorporated into the DNA chain, the DNA chain cannot be further extended. According to the principle of base pairing, whenever DNA polymerase needs dNMP to participate in the normally extended DNA chain, there are two possibilities, one is to participate in ddNTP, which results in the termination of deoxynucleotide chain extension; the other is to participate in dNTP , so that the DNA chain can still continue to extend until the next ddNTP is incorporated. According to this method, a group of DNA fragments of different lengths ending in ddNTP can be obtained. The method is to divide into four groups respectively ddAMP, ddGMP, ddCMP, and ddTMP. After the reaction, polyacrylamide gel electrophoresis can read the DNA sequence according to the swimming bands.
7. What is the positive regulation effect of activator protein (CAP) on transcription?
Cyclic adenylate (cAMP) receptor protein CRP (cAMP receptor protein), the complex formed by the combination of cAMP and CRP is called CAP (cAMPactivated protein). When E. coli is grown in a medium lacking glucose, the synthesis of CAP increases, and CAP has the function of activating promoters such as lactose (Lac). Some CRP-dependent promoters lack the typical -35 region sequence feature (TTGACA) that common promoters have. Therefore, it is difficult for RNA polymerase to bind to it. The presence of CAP (function): can significantly improve the binding constant of enzyme and promoter. It mainly shows the following two aspects: ① CAP helps the enzyme molecule to orient correctly by changing the conformation of the promoter and the interaction with the enzyme, so as to combine with the -10 region and play the role of replacing the function of the -35 region. ②CAP can also inhibit the binding of RNA polymerase to other sites in DNA, thereby increasing the probability of binding to its specific promoter.
8. What steps are usually included in a typical DNA recombination experiment?
a. Extract the target gene (or exogenous gene) of the donor organism, and enzymatically connect it to another DNA molecule (cloning vector) to form a new recombinant DNA molecule. b. Transfer the recombinant DNA molecule into the recipient cell and replicate and preserve it in the recipient cell. This process is called transformation. c. Screen and identify those recipient cells that have absorbed the recombinant DNA. d. Mass culture the cells containing the recombinant DNA to detect whether the foreign aid gene is expressed.
9. Construction of gene library Three methods for screening of recombinants are given and the process is briefly described.
Antibiotic resistance screening, insertional inactivation of resistance, blue-white spot screening or PCR screening, differential screening, DNA probe Most cloning vectors carry antibiotic resistance genes (anti-ampicillin, tetracycline). When the plasmid is transferred into Escherichia coli, the bacteria will acquire resistance, and those without transfer will not have resistance. But it cannot distinguish whether it has been reorganized or not. In a vector containing two resistance genes, if a foreign DNA fragment is inserted into one of the genes and causes the gene to be inactivated, two plate controls containing different drugs can be used to screen for positive recombinants. For example, the pUC plasmid contains the LacZ gene (encoding β-galactosidase), which can decompose the chromogenic substrate X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) to produce blue , thus turning the strain blue. When the foreign DNA is inserted, the LacZ gene cannot be expressed, and the strain is white, so as to screen the recombinant bacteria.
10. Explain the basic process of obtaining transgenic animals through embryonic stem cells?
Embryonic stem cells (ES) are embryonic cells during embryonic development, which can be artificially cultured and proliferated and have the function of differentiating into other types of cells. Culture of ES cells: The inner cell mass of the blastocyst is isolated and cultured. When ES is cultured in a feeder-free layer, it will differentiate into various functional cells such as muscle cells and N cells. When cultured in a medium containing fibroblasts, ES will maintain the differentiation function. ES can be genetically manipulated, and its differentiation function can be integrated without affecting its differentiation function, which solves the problem of random integration. Introduce exogenous genes into embryonic stem cells, then implant into the uterus of pregnant female mice, develop into pups, and cross to obtain homozygous mice.