Section 1 Tool Enzymes
The basic technology of genetic engineering is to manually cut, splice and combine genes. A gene is a DNA molecule with a certain function. To accurately cut out DNA linear molecular fragments of different genes, various restriction endonucleases are required; to connect different fragments, DNA ligase ligase); To bind a gene or one of its fragments, DNase (DNa polymerase) is required. Therefore, enzymes are indispensable tools in DNA recombination technology, and the enzymes used in genetic engineering are collectively called tool enzymes.
1. DNA restriction enzyme
Lurva and Human (1952) and Bertani and Weigle (1953) discovered the limiting effect of bacteriophage lambda, that is, a lambda bacteriophage grows well in one host cell, but grows poorly in another host cell. The reason is that Its DNA is "restricted" by the latter host. From this, a restriction-modification system was discovered.
Various bacteria can synthesize one or several endonucleases in a specific sequence. These enzymes cut the double-stranded DNA, because their function is to cut DNA, restricting the presence of foreign DNA in their own cells, so this endonuclease is called a restriction enzyme. The DNA of the cell that synthesizes the restriction enzyme is not affected by this enzyme, because the cell also synthesizes a modified enzyme, which changes the structure of the DNA sequence recognized by the restriction enzyme, so that the restriction enzyme cannot function. Restriction-modification systems are a means of defense for cells. If a bacteriophage is used to infect bacteria that are active in the restriction-modification system, the bacteriophage DNA is not modified first. Compared with the first modified bacteriophage, the infection efficiency is several orders of magnitude lower. Unmodified bacteriophage DNA is cut into fragments by restriction enzymes after entering the cell. The number of fragments is proportional to the number of restriction enzyme recognition points in the DNA molecule. These fragments are further degraded by the cell's exonuclease and will begin to lyse and infect. The resulting progeny phages all carry modified DNA, so they can infect other bacteria with the same restriction-modification system with high efficiency. At present, there are more than 175 restriction enzymes isolated from various organisms, of which more than 80 are for cutting double-stranded DNA.
(1) Naming principle
Restriction enzymes are mainly extracted from prokaryotes. Now the general naming principle is: the first word is the first letter of the bacterial genus name, the second and third words are the first two letters of the bacterial species name, these letters are in italic letters; the next is the bacterial strain The first letter is written in regular letters. If there are several different endonucleases in the same strain, they are represented by Roman numerals I, II, III ... The list is now illustrated as an example (Table 23-1).
Table 23-1 Examples of naming principles for several restriction enzymes
Bacterial original name Bacterial species name Strain name Restriction enzyme name Arthrobacter Luteus Aluâ… Bacillus amyloliquefaciens H BamHâ… Escherichia Coli RY13 EcoRâ… Haemophilus influeuzae Rd Hindâ…¢(2) Classification and characteristics
Restriction enzymes are mainly divided into three categories. The first type of restriction endonuclease can recognize a specific nucleotide sequence and cut double-strands in the DNA molecule at some nucleotides near the recognition point, but the sequence of cleaved nucleotides is not specific, is Random. Such restriction enzymes are of little use in DNA recombination technology or genetic engineering and cannot be used to analyze DNA structure or clone genes. Such enzymes as EcoB, EcoK and so on.
The second type of restriction enzyme can recognize a specific nucleotide sequence and cut the double strand at a fixed position within the sequence. Because of the restriction endonuclease recognition and cleavage of nucleotides are specific. Therefore, DNA fragments with the same nucleotide sequence can always be obtained, and DNA fragments from different genomes can be constructed to form hybrid DNA molecules. Therefore, this restriction enzyme is one of the most commonly used tool enzymes in DNA recombination technology. The specific nucleotide sequence recognized by this enzyme is most commonly 4 or 6 nucleotides, and a few recognize 5 nucleotides as well as 7, 9, 10, and 11 nucleotides. If the recognition positions are randomly distributed in the DNA molecule, the restriction enzyme that recognizes 4 nucleotides has a cut point every 46 (4096) nucleotides. The human haploid genome is estimated to be 3 × 199 nucleotides. Recognition of 4 nucleotide restriction enzymes will have (3 × 109 / 2.5 × 102) about 107 cutpoints, which is It can be cut into 107 fragments by this enzyme, and the restriction enzyme that recognizes 6 nucleotides will also have (3 × 109/4 × 103) about 106 cut points.
The recognition sequence of the second type of restriction enzymes is a palindrome symmetry sequence, that is, there is a central axis of symmetry, and "reading" from this axis in both directions is exactly the same. This enzyme can be cleaved in two ways. One is staggered cutting, resulting in two single-stranded ends. The nucleotide sequence of this end is complementary and can form hydrogen bonds, so it is called a sticky end. For example, the recognition order of EcoRI is:
↓ |
5 '... GAA | TTC ... 3'
3 '…… CTT | AAG …… 5'
| ↑
The vertical dashed line indicates the central axis of symmetry, and the nucleotide sequence "read" from both sides is either GAATTC or CTTAAG, which is the palindrome. The solid line shear indicates the position of the staggered cut on the double strand, and after the cutting, two DNA fragments, 5 '... G and AATTC ... 3', 3 '... CTTAA and G ... 5', each with a single strand At the end, the two single strands are complementary and can be "bonded" by forming hydrogen bonds. The other is to cut the double strands at the same position, resulting in blunt ends. For example, the recognition position of Haeâ…¢ is:
↓
5 '…… GG ↓ CC …… 3'
3 '... CC ↓ GG ...'
↑
Cutting at the arrow, the two DNA fragments produced are:
5 '... GG CC ... 3'
with
3 '... CC GG ... 5'
Sometimes the recognition nucleotide sequence and cleavage position of the two restriction enzymes are the same, the only difference is that when there are methylated nucleotides in the recognition sequence, one restriction enzyme can cleave, the other Species cannot. For example, the recognition order of Hpaâ…¡ and Mspâ… are both 5 '... GCGG ... 3'. If there is 5'-methylcytosine in it, only Hpaâ…¡ can cleave. These enzymes with the same cut point are called isozymes or isoschizomers.
The third type of restriction enzyme also has a specific recognition sequence, but it is not a symmetric palindrome sequence. It cuts the double strand at a fixed position of several nucleotide pairs next to the recognition sequence. But these nucleotide pairs are arbitrary. Therefore, the DNA fragments of a certain length produced by this restriction enzyme have various single-stranded ends. This is not very useful for cloning genes or cloning DNA fragments.
Second, methylase
Cell restriction-modification system modification is performed by methylase (methylase). Methylases have the same recognition sequence as restriction enzymes. Methylase methylates a certain base in the recognition sequence, protecting DNA from being cut by restriction enzymes.
Only 5-methylcytosine (M5C) is currently found in eukaryotes. M5C accounts for 2 to 7% of the entire cytosine (except for fruit flies and some insects). M5C mostly exists in the form of M5CpG. In different species or different tissues of the same species, the frequency of occurrence of M5C varies. The methylases that have been purified and sold as commercial products are shown in Table 23-2.
Simultaneous action of restriction enzymes and methylases can make several restriction enzymes that can recognize sequences effective for only one of them. For example, the recognition sequence of restriction enzyme Avaâ… is:
5 '... CPyCGPuG ... 3'
3 '... GPuGCPyC ... 5'
Py can be any kind of pyrimidine and pu can be any kind of purine. So there can be 4 kinds of recognition order. If the methylase Taq I and the methylase Hpa II are used at the same time, the recognition order of Ava I will be only 5 '... CCCGAG ... 3'. The illustration is used now (Figure 23-1).
If there is one DNA fragment, there are 3 recognition sequences of Avaâ… , and 4 fragments can be obtained after treatment with Avaâ… . However, if you first use Taq I methylase and Hpa II methylase to methylate some bases in the DNA sequence, and then use Ava I to cut, the result is that only one recognition sequence can be affected by Ava I and only two DNA fragments.
Figure 23-1 Several restriction enzymes and methylated bases
Table 23-2 Methylases and methylated bases
Methylase name
Methylated base
Aluâ… 5 '…… AGC * T …… 3' BamHâ… 5 '…… GGATC * C …… 3' Claâ… 5 '…… ATCGA * T …… 3' damâ… 5 '…… GA * TC …… 3' EcoRâ… 5 '…… GAA * TTC …… 3' Haeâ…¢ 5 '…… GCC * C …… 3' Hhaâ… 5 '…… GC * GC …… 3' Hpaâ…¡ 5 '…… CC * GC …… 3' Hphâ… 5 '… … TC * ACC …… 3 'Mspâ… 5' …… C * CGG …… 3 'Pstâ… 5' …… CTGCA * C …… 3 'Taqâ… 5' …… TCGA * …… 3 '* Indicates methylation
3. Other enzymes for molecular cloning
(1) DNA polymerase
The role of (DNa polymerase) is to add a deoxytriphosphate nucleotide to the 3'-OH of the primer to release a pyrophosphate molecule (ppi), as shown in the following formula.
There are the following polymerases:
1. E. coli DNA polymerase â… E. coli DNA polymerase (E.Coli DNA polymerase) has three main functions: â‘ 5 '→ 3' polymerization. But instead of duplicating the chromosomes, it repairs the DNA, filling in the gaps in the DNA or the gaps left after excising the RNA primers. â‘¡3 '→ 5' exonuclease activity. Eliminate false nucleotides incorporated in polymerization. â‘¢ 5 '→ 3' exonuclease activity. Excise the damaged DNA. Its application in nick translation will be described below.
The Klenow fragment of E. coli DNA polymerase I is a complete DNA polymerase I fragment, only 5 '→ 3 polymerase activity and 3' → 5 'exonuclease activity, lost 5' → 3 exonuclease activity . It can be used to fill single-stranded DNA ends into double strands. If 32P-labeled nucleotide triphosphate is supplied, the DNA can be labeled with an isotopic label. When the DNA fragments with single-stranded cohesive ends are cut with interleaved restriction enzymes, and the DNA fragments with blunt ends are to be ligated, the single-stranded ends of the cohesive ends can be blunt-ended with Klenow fragments, and DNA ligase connects two DNA fragments.
There are also E. coli DNA polymerase II and E. coli DNA polymerase III. The former cannot use single-stranded DNA or poly (dA-dT) as a template. Magnesium ions and dNTPs are required to exhibit enzyme activity without the effect of spontaneously synthesizing DNA. The latter does not have 5 '→ 3 exonuclease activity, nor can it use single-stranded DNA as a template.
2. Phage DNA polymerase here takes T4 DNA polymerase as an example. It also has 5 '→ 3 polymerase activity, but its exonuclease activity is 200 times higher than that of E. coli. Therefore, it can also be used to fill single-strand ends or to label isotopes.
(2) RNA polymerase
The role of RNA polymerase (RNa polymerase) is to transcribe RNA. Some RNA polymerases have relatively complex subunit structures. For example, E. coli RNA polymerase has four polypeptide chains, and there is another σ factor that promotes the synthesis of new RNA molecules, so it is composed of α2ββσ. This structure is called holoenzyme, and the enzyme with the σ factor removed is called the core enzyme. Phage RNA polymerase has no subunits.
There are three types of RNA polymerases in eukaryotes. RNA polymerase I exists in the nucleolus and transcribes rRNA sequences. RNA polymerase II exists in the nucleoplasm and transcribes most genes, requiring a "TATA" box. RNA polymerase III exists in the nucleoplasm and transcribes few genes such as tRNA genes such as 5SrRNA genes. Some repetitive sequences such as Alu sequence may also be transcribed by this enzyme. The "TATA" box mentioned above, also known as the Goldberg-Hogness sequence, is the contact point of RNA polymerase II and is unique to the transcription unit of this enzyme. It is on the 5 'end of the transcribed genes of eukaryotes, and has an AT-rich sequence between 20 and 30 nucleotides upstream of the transcription start point. If the transcription start point is 0, there is a "TATA" box between -33 to 27 nucleotides and -27 to 21 nucleotides. Generally 7 nucleotides. The typical structure of RNA polymerase II transcription unit is shown in Figure 23-2. Prokaryotes are also similar to the "TATA" box structure. RNA polymerase acts near the "TATAAT" (Pribnow) box and the "TTGA-CA" box.
Figure 23-2 Mechanism of action of reverse transcriptase
(3) Reverse transcriptase
Reverse transcriptase (Reverse transcripatase) is an enzyme that uses RNA as a template to guide the synthesis of complementary DNA (cDNA) by deoxynucleotide triphosphate. The reverse transcriptase of mammalian type C virus and the reverse transcriptase of murine type B virus are both a polypeptide chain. The reverse transcriptase of avian RNA virus is composed of two upper subunits. Reverse transcriptases with different structures have also been isolated in eukaryotes. This enzyme requires magnesium ions or manganese ions as cofactors. When mRNA is used as a template, single-stranded DNA (ssDNA) is synthesized first, and then under the action of reverse transcriptase and DNA polymerase I, single-stranded DNA is used as a template "Hairpin" type double-stranded DNA (dsDNA), and then cut into two single-stranded double-stranded DNA by nuclease S1. Therefore, reverse transcriptase can be used to reverse transcribe the mRNA of any gene into a cDNA copy, and then a large amount of cDNA inserted into the vector can be amplified. It can also be used to label cDNA as a radioactive molecular probe.
(4) Exonuclease
E. coli exonuclease Ⅲ (exonuclease Ⅲ) is a double-stranded DNA with 3'-OH ends, and excises 5 'single nucleotides in the direction of 3' → 5 '
E. coli exonuclease â…¦ cuts oligonucleotides (two forms) from the 3 'and 5' ends of single-stranded DNA:
(1) (2)
The former enzyme requires magnesium ions as cofactors to be active; the latter enzyme does not require magnesium ions, so it is active even in the presence of the chelating agent EDTA. Another type of lambda exonuclease extracted from bacteriophage lambda-infected E. coli is to excise 5 'single nucleotides from double-stranded DNA with 5' phosphate ends one by one, and magnesium ions are required for the reaction:
(5) Nuclease S1
Nuclease S1 (nuclease S1) mainly degrades single-stranded DNA or single-stranded RNA. Higher activity on single-stranded DNA. Its purpose is to excise the single-stranded ends of DNA fragments into blunt ends, cut the "hairpin" loops formed during the synthesis of dscDNA, and analyze the structure of DNA-RNA heterozygotes.
(6) DNase
DNase I (DNase I) is an endonuclease that hydrolyzes double-stranded or single-stranded DNA randomly, degrading DNA molecules into a mixture of single nucleotides and oligonucleotides with 5 'phosphate ends:
When conducting recombinant DNA research, care must be taken to prevent DNase contamination, otherwise the prepared DNA samples will degrade. Therefore, utensils or reagents are treated at high temperature, and EDTA is added to the sample, or placed on ice to destroy or inhibit enzyme activity.
(7) RNase
Ribonuclease A (ribonuclease A) acts on the 3 'phosphate of pyrimidine nucleotides and cuts the 5' phosphate bond to adjacent nucleotides. Another RNase T1 acts only on the 3 'phosphate of guanine nucleotides, cutting the 5' phosphate bond to the adjacent nucleotide. Human secretions such as saliva and sweat contain RNase. Therefore, when handling RNA samples, gloves must be worn, and the glassware used in the experiment must be baked at 250 ℃ for 4h (RNAase heat resistance), or treated with RNase inhibitors.
(8) Ligase
The most commonly used is T4 DNA ligase (T4DNa ligase), which catalyzes the formation of phosphodiester bonds between adjacent 3'OH and 5 'phosphates in double-stranded DNA. It can be used to connect two DNA fragments with sticky ends, or The two blunt-end DNA fragments are joined to make it a recombinant DNA molecule. However, this enzyme can only connect double-stranded DNA but not single-stranded DNA molecules. T4RNA ligase can catalyze the formation of a covalent bond between the 5 'phosphate and 3'-OH of a single-stranded DNA molecule or RNA molecule.
(IX) Terminal transferase
The role of terminal deoxynucleotide transferase is to add deoxynucleotide to the 3'-OH end of DNA molecule.
(10) Alkaline phosphatase
The 5'-phosphate residue is removed from the deoxyribonucleotide triphosphate or ribonucleotide triphosphate of DNA or RNA. The general purpose is to label 32P on the 5 'end after treating DNA or RNA with this enzyme. After digesting the DNA fragment with a restriction enzyme, treatment with alkaline phosphatase (alkaline phosphatase) can prevent the cleaved fragment from joining itself, which is particularly useful when cloning DNA fragments.
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