Experiment 5: Polymerase Chain Reaction (PCR)

　　I. Experimental Principle

　　Polymerase chain reaction (PCR) is an in vitro nucleic acid synthesis technology for specific amplification of specific DNA fragments. This technology is a breakthrough in the field of molecular biology research.

　　PCR usually requires two oligonucleotide primers located on both sides of the fragment to be amplified. These primers are complementary and oriented to the two strands of the fragment to be amplified, allowing the region between the two primers to be amplified by polymerase. The reaction process first requires that the DNA to be amplified (called the template) be denatured into single-stranded templates at high temperature; this process is called denaturation. The second step is annealing, where artificially synthesized oligonucleotide primers (Primer 15-20bp) complementary to the 3' end of the two strands of the DNA fragment to be amplified bind to both sides of the template at low temperature. The third step is extension, where DNA polymerase extends deoxyribonucleotides (dNTP: dATP, dCTP, dTTP, dGTP) along the primer in the 5'→3' direction to synthesize new DNA strands at an appropriate temperature. Denaturation-annealing-extension, this cycle repeats, and the new DNA strands produced in each cycle can become templates for the next cycle. Therefore, PCR products are amplified exponentially by 2n. After 30-35 cycles, the target fragment can be amplified by a million times. On a typical PCR instrument, such a reaction takes several hours to complete.

　　Schematic Diagram of PCR Principle

　　II. Experimental Methods

　　1. On ice, set up the following PCR reaction system, adding separately into the PCR tube:

　　10×PCR Buffer 2μL

　　25mM dNTPS 1.6μL

　　2.5mM MgCl2 1.2μL

　　Primer 1(10μM) 0.8μL

　　Primer 2(10μM) 0.8μL

　　Template DNA (1μg/μL) 1μL

　　Taq enzyme (5U/μL) 0.2μL

　　dd H2O 12.4μL

Total volume 20μL

　　2. Place the PCR tubes into the PCR instrument and operate according to the following program:

　　①94℃ pre-denaturation 2min (initial template DNA denaturation should be appropriately extended)

　　②94℃ denaturation 1 min → 36℃ annealing 1 min → 72℃ extension 2 min, total 40 cycles

　　③72℃ extension 7 min (the time for the last extension should also be appropriately extended)

　　④4℃ storage.

　　3. Take 1-5μL of reaction product for agarose gel electrophoresis detection.

　　III. PCR Optimization

　　To achieve the desired PCR amplification effect and select the most suitable and repeatable conditions, different reaction components and cycling parameters should be tested. Factors such as Mg2+ concentration, dNTP concentration, template DNA content, and Taq enzyme content significantly affect the experimental results. In preliminary experiments, gradient experiments and interactive combination experiments should be carried out separately to finally determine the optimized PCR reaction system.

　　IV. Required Instruments and Consumables

　　1. Micropipette

　　2. Micropipette tips

　　3. PCR tubes

　　4. PCR instrument

　　5. Centrifuge

　　6. Centrifuge tubes

　　7. Glassware

　　V. Required Reagents

　　1. Random Primers (10μM)

　　2. dNTP (25mM)

　　3. Template DNA (1μg/μL)

　　4. Taq DNA Polymerase (5U/μL)

　　5. 10×PCR Buffer

　　6. TBE

　　7. Loading Buffer

　　8. MgCl2 (25mM)

　　VI. Reagent Preparation

　　1. Reagents required for agarose gel electrophoresis are the same as in Experiment 4.

　　2. Primer preparation:

　　Most ordered primers are dry powder and attached to the tube wall, making them extremely easy to lose when opened. Therefore, centrifuge before opening the tube, then slowly open the cap, dissolve and adjust the concentration, then close the cap and shake thoroughly up and down for 5-10 minutes. Store below -20℃ when not in use.

　　The dry powder tubes generally indicate the absorbance at 260nm and the number of base pairs. For example, a tube labeled 5 OD 20 mer oligo DNA means a 20bp oligonucleotide DNA with an absorbance of 5 OD at 260nm.

　　Molecular Weight (MW) = (A base count × 312) + (C base count × 288) + (G base count × 328) + (T base count × 303) - 61

　　Molecular weight can also be calculated using the following approximate method:

　　The average molecular weight of a deoxyribonucleotide base is approximately 324.5, so the molecular weight of a primer = number of bases × 324.5

　　For example: if a primer tube is labeled 0.2 OD 10 mer, to prepare a primer concentration of 10μM, the method is as follows:

　　Molecular Weight = 10 × 324.5 = 3245

　　Mass = 0.2 × 33 = 6.6μg (1 OD ssDNA = 33μg)

　　Moles = 6.6μg / 3245 = 0.002 μmoL = 2 nmoL

　　(2 nmoL/10μM)×1000=200μL

　　Therefore, 200μL of water should be added to adjust the primer in this tube to 10μM.

　　VII. Questions

　　1. What is the reaction principle of PCR?

　　2. What components does the PCR reaction system include?

　　Experiment VI Random Amplified Polymorphic DNA Reaction (RAPD)

　　I. Related Knowledge

　　Genetic markers are important tools for studying the laws of biological genetic variation and their material basis. There are mainly four types of methods: morphological markers, cytological markers, biochemical markers, and molecular markers. Compared with the first three types of markers, molecular markers have the following advantages: (1) Directly using nucleic acids as the research object, they can be detected in various tissues and developmental stages of plants, unrestricted by season or environment, unrelated to developmental stage, and can be used for early selection of plant genotypes. (2) The number of markers is extremely large, covering the entire genome. (3) High polymorphism, as many allelic variations exist naturally, requiring no specific creation of special genetic materials. (4) Many molecular markers exhibit codominance, enabling the differentiation of homozygous and heterozygous genotypes, providing complete genetic information.

　　Currently, commonly used molecular marker techniques mainly include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), chromosome or genomic in situ hybridization (GISH), mRNA differential display (mRNA-DD), suppression subtractive hybridization (SSH), subtractive hybridization (SH), representation difference analysis (RDA), arbitrary-primer PCR (AP-PCR), DNA amplified fingerprinting (DAF), single primer amplification reaction (SPAR), sequenced characterized amplified region (SCAR), anchored microsatellite oligonucleotide (AMO), single nucleotide polymorphism (SNP), sequence-tagged site (STS), and so on.

　　I. Experimental Principle

RAPD technology is based on PCR technology. It uses a series (usually hundreds) of oligonucleotides (typically decamers) with randomly arranged base sequences as primers. After the template is denatured and unwound at 92-94°C, if the double-stranded DNA molecule has reverse parallel fragments complementary to the primer within a certain length, and the primer positions are within amplifiable distance from each other (primer spacing is 200-2000bp), then discontinuous DNA fragments with molecular weights of 200-2000bp will be produced through PCR. High temperature denaturation, low temperature annealing, and moderate extension are repeated for 30-40 cycles, after which the product can be amplified by more than a million-fold. Such high-yield DNA fragments are separated by electrophoresis and stained with EB, then directly observed under UV light and photographed.

　　RAPD technology is simple and easy to master, allowing for the rapid acquisition of a large number of genetic markers in a short period. As these advantages have gradually gained acceptance, it has developed rapidly, finding application in numerous biological fields within just three years, such as genetic fingerprinting, mapping, and detecting genetic diversity and stability (variety identification, origin and evolution of species), etc.

　　III. Experimental Methods

　　(I) DNA Extraction

　　Same as the previous CTAB method.

　　(II) Purity Detection and Concentration Adjustment

　　Same as Experiment IV.

　　(III) Operating Procedure

　　1. Preparation of Reaction System

　　Prepare the following reaction system on ice. Take a PCR tube and add the following components:

　　10×PCR Buffer 2.0 µl

　　模板DNA(500ng/µl) 1.0 µl

　　dNTPs(2.5mM) 1.6 µl

　　MgCl2 (25mM) 2.0 µl

　　Primer(20µM) 1.2 µl

　　Taq酶(2.5U/µl) 0.5 µl

　　Add ddH2O to total volume 20.0 µl

　　Mix well by pipetting up and down, then centrifuge to collect. Seal with mineral oil.

　　2. Amplification Program

　　Then place the PCR tubes into the PCR instrument and operate according to the following program.

　　94℃，预变性5min;94℃变性1min，37℃退火1min，72℃延伸2min，40个循环;72℃延伸7min，4℃保存。

　　3. Agarose Gel Electrophoresis Detection

　　Method same as Experiment III, gel concentration is 2%.

　　IV. Factors Affecting the Reaction and Precautions

　　(I) Influencing Factors

　　During the RAPD reaction, many reaction factors are involved, and they are sensitive to reaction conditions. Improper setting of any reaction factor will affect the entire reaction process or alter the band pattern. Generally, laboratories need to systematically explore various reaction conditions and influencing factors to optimize the reaction system and obtain a standardized RAPD reaction under experimental conditions.

　　The influencing factors during the reaction mainly include Taq enzyme, Mg2+ concentration, amplification reaction temperature and time, cycle number, primers, dNTPs concentration, and DNA concentration and purity. Therefore, the reaction system and amplification conditions must be fully optimized before the experiment.

　　(II) Precautions

　　1. Prepare the PCR reaction solution on ice, then place it on the PCR instrument for reaction.

　　2. High DNA quality is required; it should be purified and special attention paid to preventing oxidation.

　　3. The reaction is sensitive and prone to contamination, leading to false positives, hence control reactions are necessary.

　　4. Pay attention to influencing factors and the occurrence of the "plateau effect".

　　5. If the product specificity is low, 5% dimethyl sulfoxide (DMSO) or similar can be added to the reaction mixture to increase product specificity.

　　6. If the product resolution on agarose gel is low, polyacrylamide gel or gradient gel electrophoresis can be used to improve resolution.

　　V. Instruments and Consumables Used

　　PCR machine; micropipette; centrifuge; UV analyzer; electrophoresis apparatus; PCR tubes; sterile pipette tips; glassware, etc.

　　VI. Reagents Required

　　10×PCR Buffer; template DNA; dNTPs (2.5mM); MgCl2 (25mM); random primers (20µM); Taq enzyme (2.5U/µl); loading buffer; agarose, etc.

　　VII. Questions

　　1. Discuss the differences between RAPD technology and PCR technology.

　　2. Discuss why RAPD technology has poor repeatability?

　　Experiment Seven: AFLP Analysis of Sugar Beet M14 Line

　　I. Related Knowledge

　　(1) Genomic DNA Restriction Endonuclease Digestion

　　In genetic engineering technology, enzyme digestion is a critical operational process. Appropriate restriction endonucleases are selected to cut the vector into desired fragments for the insertion of exogenous genes. Endonucleases are enzymes that cut inside DNA strands. Endonucleases are divided into two categories: non-restriction endonucleases and restriction endonucleases. Non-restriction endonucleases refer to those endonucleases that do not recognize specific DNA sequences for cutting, such as deoxyribonuclease I (DNase I), which is an endonuclease extracted from bovine pancreas. DNase I is sensitive to both single- and double-stranded DNA. It can cleave phosphodiester bonds at any position within the DNA strand without a specific recognition sequence. Generally speaking, a DNA system treated with DNase I enzymatic hydrolysis will ultimately only contain degraded mononucleotides and very short oligonucleotide chains.

　　Restriction endonucleases, on the other hand, are the opposite of non-restriction endonucleases. They all recognize specific DNA sequences and cleave DNA strands under certain conditions. Restriction endonucleases are divided into three types: Type I restriction endonucleases recognize DNA sequences about a dozen nucleotides long, or are methylated, or cut DNA approximately 1000bp away from one end of the recognition sequence. Type I restriction endonucleases specifically recognize DNA sequences, but their cleavage sites are not specific. Type III restriction endonucleases are somewhat different from Type I restriction endonucleases; they can also recognize specific DNA sequences, but their DNA cleavage sites are often very close to the recognition binding site, not as far as 1000bp. Nevertheless, their cleavage sites are still non-specific. Therefore, Type I and Type III restriction endonucleases do not have broad application prospects in gene manipulation technology. Type II restriction endonucleases not only specifically recognize DNA sequences, but also have their recognition DNA sequences consistent with the enzyme's DNA cleavage positions. This avoids the uncertainty and reproducibility of enzymatic cleavage ends, thus being widely used in gene recombination technology. Most restriction endonucleases do not cut at the same position on both DNA strands; instead, the two strands are broken two to four nucleotides apart, resulting in DNA ends with a 5′ overhang or a 3′ overhang single-stranded DNA. These ends are called sticky ends.

　　Enzyme digestion usually involves selecting different digestion methods based on the operational purpose, such as single digestion, double digestion, partial digestion, and so on. Single digestion is the most common method for DNA fragmentation, using one restriction endonuclease to cut the DNA sample. For double digestion, two restriction endonucleases can sequentially cut the DNA sample in different reaction systems. Using this method, the enzyme requiring a lower salt concentration buffer should be used first for cutting, then the buffer's salt concentration is adjusted, and finally the enzyme requiring a higher salt concentration buffer is added for cutting. If the optimal reaction temperatures of the two restriction endonucleases are different, the enzyme with the lower optimal reaction temperature should be used first for cutting, and then the second enzyme added after increasing the temperature. If the reaction systems for the two restriction endonucleases differ significantly, which would obviously affect the double digestion result, the desired DNA fragments can be recovered by gel electrophoresis after the first enzyme digestion, and then an appropriate reaction system can be selected for the second restriction endonuclease digestion.

　　(2) Ligation of Sticky Ends

　　In cells, the enzymes that perform ligation are ligases. These are a very special class of enzymes that catalyze the formation of phosphodiester bonds between adjacent 5′ phosphate and 3′ hydroxyl ends of DNA or RNA, thereby sealing DNA single-strand nicks. Commonly used ligases mainly include DNA ligase and RNA ligase, whose main functions are discontinuous repair and ligation.

　　Theoretically, the optimal temperature for ligation reaction is 37℃, as ligase activity is highest at 37℃. However, in actual experiments, the pairing structures formed by sticky-end DNA molecules are extremely unstable at 37℃. Therefore, researchers need to find an optimal temperature that both maximizes ligase activity and helps stabilize transient pairing structures. Currently, the commonly used ligation temperature is 12-16℃.

　　Because sticky ends have higher ligation efficiency than blunt ends, sticky-end ligation is often preferred during DNA recombination and ligation experiments. However, in reality, not all ligations have suitable sticky ends; for example, in most cases, one molecule is a sticky-end molecule and the other is blunt-end DNA, or the two molecules have different sticky ends, and so on. In these situations, methods such as linker technology, adaptor technology, and homo polymer tailing technology can be employed.

　　(3) Selective PCR Amplification

　　The principle is exactly the same as standard PCR; the only difference lies in the primers used for PCR. Primers for standard PCR are nucleotide sequences complementary to known sequences. In selective amplification, for the unknown nucleotide sequence in the middle, 1-3 random bases (ensuring a certain GC content) are artificially added after the known sequence primers. This way, only fragments that complementarily pair with these three random bases can be amplified from a large number of fragments, thereby allowing the control of the number of amplified fragments based on the number of selective bases, achieving the purpose of selectivity.

　　Primer composition: (1) Core base sequence, which is complementary to the synthetic adapter; (2) Restriction endonuclease recognition sequence; (3) Selective bases at the 3′ end of the primer.

　　(IV) Polyacrylamide Gel Electrophoresis

　　Based on the material used to prepare the gel, gel electrophoresis can be divided into two subclasses: agarose gel electrophoresis and polyacrylamide gel electrophoresis. Polyacrylamide separates small DNA/RNA fragments (5-500bp) with extremely high resolution; DNA fragments differing by just 1bp can be separated. Its disadvantages are that preparation and operation are relatively difficult. The resolution of agarose gel is lower than that of polyacrylamide gel, but its separation range is wider. Agarose gels of various concentrations can separate DNA/RNA fragments ranging from 200bp to nearly 50kb in length.

　　Commonly used polyacrylamide gels include the following two types:

　　(1) Non-denaturing polyacrylamide gel for separating and purifying double-stranded DNA fragments

　　The migration rate of most double-stranded DNA in non-denaturing polyacrylamide gel is roughly inversely proportional to the logarithm (base 10) of its size, but the migration rate is also affected by its base composition and sequence, so that DNA of exactly the same size can have migration rates differing by up to 10%. This effect is caused by the formation of kinks in double-stranded DNA at specific sequences.

　　(2) Denaturing polyacrylamide gel for separating and purifying single-stranded DNA

　　These gels are polymerized in the presence of a reagent that inhibits base pairing in nucleic acids (such as urea or formaldehyde). The migration rate of denatured DNA in these gels is almost entirely independent of its base composition and sequence. Denaturing polyacrylamide gels are used for the separation of radioactive DNA probes, the analysis of S1 nuclease digestion products, and the analysis of DNA sequencing reaction products.

　　(V) AFLP Technology

　　1. Advantages of AFLP Technology

　　Theoretically, due to the large number and variety of restriction endonucleases and selective bases used in AFLP, the number of markers that can be generated by AFLP is infinite. In AFLP analysis of genotypes, the number of bands detected by non-denaturing PAGE electrophoresis per reaction product is between 50 and 100, making this technique a very useful tool for DNA polymorphism detection. AFLP markers are typically inherited in a Mendelian fashion. Most amplified fragments in AFLP analysis correspond to a single genomic locus, thus AFLP markers can be used as landmarks for genetic and physical mapping. AFLP can be used to analyze genomic DNA of varying complexity, as well as cloned large DNA fragments, making it not only a DNA fingerprinting technique but also a very useful tool for genomic research. Random amplification of DNA is greatly affected by template concentration, whereas a significant feature of AFLP is its insensitivity to changes in template concentration. VOs et al. found that in AFLP analysis of tomatoes, results were largely consistent within a 1000-fold difference in template concentration. Only when template concentration was very low were bands weaker or even missing. Another important feature of AFLP technology is that during the reaction, all labeled primers are consumed. Once primers are consumed, the amplification band pattern is not affected by the number of cycles. Because AFLP is insensitive to template concentration, using excess cycles ensures that even with some differences in template concentration, bands of consistent intensity are obtained. Therefore, AFLP technology can detect polymorphism in band intensity.

　　2. Comparison of AFLP with Other Molecular Marker Technologies

　　Every molecular marker has its advantages and disadvantages. For the same material, the polymorphism revealed by different molecular markers varies. Generally, AFLP has the highest polymorphism ratio, followed by RFLP and RAPD. Wang et al. reported in their study on temperature-sensitive male sterile alleles in rice that the polymorphism ratios of the three molecular markers were AFLP > RAPD > RFLP (26.67%; 4.00%; 1.67%) respectively. Concurrently, Lin et al. also demonstrated the highest polymorphism for AFLP in their soybean analysis.

　　Characteristic RFLP RAPD AFLP

Distribution Widespread Widespread Widespread

　　Reliability High Medium High

　　Reproducibility High Medium High

　　Inheritance Co-dominant Dominant Dominant or Co-dominant

　　Polymorphism Medium High Very High

　　DNA Requirement 2-30ng 1-100ng 100ng

　　Radioactivity Generally Present Absent Present or Absent

　　Technical Difficulty Medium Simple Medium

　　Sample Throughput Medium-Low High Very High

Time Factor Long Fast Medium

　　Compared to other molecular markers, AFLP's main characteristics are that it combines the advantages of both RFLP and RAPD. It is convenient and fast, requires only a very small amount of DNA material, does not require Southern hybridization, does not require prior knowledge of DNA sequence information, and produces stable and reliable experimental results, allowing for rapid acquisition of large amounts of information. Furthermore, it has high reproducibility and good repeatability, making it very suitable for cultivar fingerprinting, genetic linkage map construction, and genetic diversity studies. In cases where polymorphism is low and the number of samples to be tested is small, AFLP analysis can achieve satisfactory results. For constructing high-density genetic maps or fine-mapping a specific gene region, AFLP is a relatively ideal method. The polymorphism generated by AFLP far exceeds that of RFLP, RAPD, etc., and it is currently considered the most polymorphic technique among DNA fingerprinting technologies.

　　3. Applications of AFLP Technology

　　Since its inception, AFLP technology has been widely used in genomic analysis of organisms, such as resistance gene mapping and chromosome mapping in plants, and phylogenetic analysis of pathogens. Due to its good resolution for subtle genomic changes, it has also been widely used for analyzing the affinity groups of vegetatively compatible strains of the same pathogenic fungus, and for analyzing avirulence genes and virulence of pathogens.

　　① Research on the application of AFLP technology in bacterial classification and identification

　　The reproducibility of AFLP is stronger than RAPD, and its applicability in bacterial classification is similar to RAPD, RFLP, and bacterial soluble protein profiles. However, AFLP combines the advantages of the above methods, with resolution only lower than whole-genome sequence analysis, strong stability, and the ability to provide richer classification information.

　　② Research on the application of AFLP technology in fungal classification

　　AFLP is a powerful new technique for representing the molecular characteristics of fungi. Its operation process is convenient, and its differentiation and identification results are reliable. This technique's application in pathogenic fungi has become very prominent, and some practical procedures have been developed to distinguish specific strains for typing, identification, and kinship studies. It can analyze differences between different species and even reveal subtle variations among strains within the same species, thereby compensating for the shortcomings of phenotypic typing and more accurately analyzing the essential differences of pathogenic fungi. This technique has been applied to the identification and differentiation of different source isolates of Fusarmiu graminearum, with good results.

　　II. Experimental Principle

　　In 1991, Gustava et al. successfully amplified human, animal, plant, fungal, bacterial, and viral DNA using very short oligonucleotide fragments of 5, 8, and 10 bases as primers, which they termed Amplification Fragment Length Polymorphisms (AFLP). The AFLP technique established by Zabeau and Vos in 1992 is significantly different from the aforementioned. This method combines the characteristics of RFLP (restriction fragment length polymorphisms) and RAPD techniques. It uses artificial adaptors (Adaptor) to ligate with genomic DNA fragments digested by restriction endonucleases, using this as a DNA template to synthesize a series of PCR primers with several randomly varied 3'-terminal bases complementary to the artificial adaptor sequence. These primers are then used for PCR amplification under specific conditions, and DNA fingerprint maps can be obtained after electrophoresis detection.

　　Genomic DNA

　　CTGCAGNNNN…NNNNCTGCAG…

　　GACGTCNNNN…NNNNGACGTC

PstⅠ Restriction Digestion

　　GNNNN…NNNNCTGCA

　　ACGTCNNNN…NNNNG

Add Artificial Adaptor

　　5’---CTCGTAGACTGCGTACATGCA---3’

　　3’--- CATCTGACGCATGT ---5’

T4 Ligase Ligation

　　lCTCGTAGACTGCGTACA TGCA GNNNN…NNNNCTGCA

　　CATCTGACGCATGT ACGT CNNNN…NNNNG

Select Primer PCR Amplification

　　CTCGTAGACTGCGTACATGCAGNNNN…NNNNCTGCA

　　CATCTGACGCATGTACGTCNNNN…NNNNG

　　Ps1--GACTGCGTACATGCAGACC

　　Ps2—GACTGCGTACATGCAGCTG

　　Gel Detection

　　(1) Genomic DNA Restriction Endonuclease Digestion

　　The efficiency of restriction endonucleases is influenced by various factors, such as reaction temperature, buffer system, ion type and concentration, DNA purity, DNA molecule methylation level, etc. The reaction buffer for restriction endonucleases generally contains MgCl2, NaCl or KCl, Tris-HCl, dithiothreitol (DTT) or β-mercaptoethanol, and some also contain bovine serum albumin (BSA). Mg2+ is a cofactor for restriction endonucleases, and Tris-HCl maintains the pH of the entire reaction system; different enzymes have different requirements for Na+ or K+, and DTT and β-mercaptoethanol help stabilize the enzyme in the system.

　　The purity of DNA significantly affects the restriction digestion results, as impurities like proteins, phenol, chloroform, and SDS can directly inhibit enzyme activity after contaminating the DNA. During experiments, to overcome the influence of these impurities, measures taken include extending the reaction time, increasing the enzyme amount, or both increasing the enzyme amount and extending the reaction time to achieve good reaction results.

　　Different restriction endonucleases may have different optimal reaction temperatures. The standard reaction temperature for endonucleases is 37℃, but there are many exceptions, such as Sma I's optimal reaction temperature being 25℃, and Taq I's optimal reaction temperature being 65℃. The definition of endonuclease activity: one unit of activity is defined as the amount of enzyme that completely digests 1μg of λDNA in 50 μL of reaction solution at the appropriate temperature in 1 hour.

　　Restriction endonuclease stock solutions usually contain 50% glycerol. When glycerol exceeds 5% in the reaction system, it will affect the specificity of restriction digestion. Therefore, the volume of enzyme added during restriction digestion should not exceed 1/10 of the total reaction volume.

　　The restriction digestion time varies depending on the experiment and is determined by the concentration and purity of the DNA sample and the enzyme concentration. High DNA sample concentration, poor purity, or too low enzyme concentration can all warrant an appropriate extension of the digestion time. The volume of the restriction digestion system is generally suitable at 20-50 μL. If the DNA sample is genomic DNA, the time can be extended to 18 hours or longer.

　　EcoR Ⅰ:

5′----GAATTC---3′ 5′---G AATTC---3′

3′----CTTAAG---5′ 3′---CTTAA G---5′

　　Pst Ⅰ:

5′---CTGCAG---3′ 5′---CTGCA G---3′

3′---GACGTC---5′ 3′---G ACGTC---5′

　　BamH Ⅰ:

5′---GGATCC---3′ 5′---G GATCC---3′

3′---CCTAGG---5′ 3′---CCTAG G---5′

　　(II) Adaptor Ligation

　　(3) Selective PCR Amplification

　　(IV) Polyacrylamide Gel Electrophoresis

　　Polyacrylamide gel (polyacrylamide gel, PAG) is polymerized from acrylamide and the cross-linking agent N,N'-methylenebisacrylamide in the presence of initiators such as ammonium persulfate and accelerators such as N,N,N',N'-tetramethylethylenediamine (TEMED). Acrylamide monomers form long chains, and cross-linking occurs through the reaction of the bifunctional groups of N,N'-methylenebisacrylamide and the free functional groups at the chain ends, forming a gel. The pore size of the gel is determined by the chain length and degree of cross-linking.

　　The preparation and electrophoresis of polyacrylamide gels are more cumbersome than those of agarose gels. Polyacrylamide gels are almost always cast between two glass plates, separated by spacers and sealed with insulating tape. In this configuration, most acrylamide solution does not come into contact with air, so oxygen inhibition of polymerization is limited to a narrow layer at the top of the gel. Polyacrylamide gels are always run by vertical electrophoresis, and their length can range from 10 to 100 cm depending on the separation requirements. Polyacrylamide gels have three main advantages compared to agarose gels: ① High resolving power, capable of separating DNA molecules differing in length by only 0.2% (i.e., 1 bp out of 500 bp); ② The amount of DNA that can be loaded is much greater than for agarose gels; up to 10 μg of DNA can be loaded into a standard sample well of a polyacrylamide gel without significantly affecting resolution; ③ DNA recovered from polyacrylamide gels is of high purity and suitable for the most demanding experiments.

　　III. Experimental Methods

　　(1) Genomic DNA Restriction Endonuclease Digestion

　　1. The DNA used in this experiment was genomic DNA extracted from beet leaves. Three restriction endonucleases were selected for single enzyme digestion reactions: Pst I, BamH I, and EcoR I.

　　2. Take three 0.5mL centrifuge tubes, add the following components in order. The total reaction volume is 20μL.

Sterile water 16μL

　　10× Restriction Endonuclease Buffer 2 μL

　　Genomic DNA(1mg/mL) 2 μL

　　Restriction Endonuclease 1 μL

　　3. Mix by pipetting up and down, centrifuge briefly to collect all liquid at the bottom of the tube. Place the centrifuge tubes in a water bath and incubate for 2.5–4 h according to the optimal temperature of different enzymes.

　　4. Inactivate in a 65℃ water bath for 15 minutes to terminate the enzyme digestion reaction.

　　5. Take 10 μL of the digested solution, add 2 μL of 6× loading buffer, and load onto a 1% agarose gel for electrophoresis. Use a molecular weight Marker as a size control during electrophoresis. The electrophoresis current is 60–100mA.

　　6. After electrophoresis, observe under UV light. Completely digested total DNA should appear as a uniform smear in the lane, with brighter bands often visible. These are repetitive sequences.

　　(II) Adaptor Ligation

　　1. Each restriction endonuclease has its corresponding adaptor. The sequences are as follows:

　　EcoR Ⅰ:

　　Ead1： 5′--CTC GTA GAC TGC GTA CC--3′

　　Ead2： 3′--CAT CTG ACG CAT GGT TAA--5′

　　Pst Ⅰ：

　　Pad1： 5′--CTC GTA GAC TGC GTA CAT GCA--3′

　　Pad2： 3′--CAT CTG ACG CAT GT--5′

　　BamH Ⅰ：

　　Bad1： 5′--GGG TCG AAT TCG AGC TCA G--3′

　　Bad2： 3′--CCC AGC TTA AGC TCG AGT CCT AG--5′

　　2. Preparation of adaptors:

　　① First, prepare each single-stranded part of the adaptor as a 50μM solution, calculate the amount of water to add based on the molar quantity;

　　② Take 25μL of solution from each of the two tubes and add to a PCR tube. After denaturation at 94℃ for 2 min and annealing at 36℃ for 5 min, allow to cool naturally to room temperature. The two complementary oligonucleotide strands will then combine to form an artificial adaptor. Label and store for later use.

　　3. Preparation of ligation reaction system:

　　Take one PCR tube, add the following components sequentially on ice:

Digested template DNA 250 ng (5 μL)

　　25μM Adaptor 0.2 μL

　　T4 Ligation Buffer 5 μL

　　T4 Ligase(3U/μL) 0.6 μL

　　ddH2O 39.2 μL

Total volume 50 μL

　　4. Mix by pipetting. Incubate at 16℃ O/N (overnight) for ligation reaction.

　　5. Add 0.1× TE Buffer to a final volume of 250 μL, store at 4℃ for later use.

　　(3) Selective PCR Amplification

　　1. The selective amplification primers used for the three restriction endonucleases. Their sequences are as follows:

　　EcoR Ⅰ:

　　Es1： 5′--GAC TGC GTA CCA ATT CGT C--3′

　　Es2： 3′--GAC TGC GTA CCA ATT CAG C--5′

　　Pst Ⅰ:

　　Ps1： 5′--GAC TGC GTA CAT GCA GCT G--3′

　　Ps2： 3′--GAC TGC GTA CAT GCA GAC C--5′

　　BamH Ⅰ:

　　Bs1： 5′--TTC GAG CTC AGG ATC CGT G--3′

　　Bs2： 3′-- GAG CTC AGG ATC CAC G--5′

　　2. Primer preparation:

　　Before use, prepare primers to a final concentration of 66 ng/μL according to molarity, for later use.

　　3. Preparation of PCR reaction system:

　　Take one PCR tube, add the following components sequentially on ice:

10×PCR Buffer 2.0 μL

　　2.5 mM dNTP 1.6 μL

　　25 mM MgCl2 1.2 μL

　　Selected Primer 1.0 μL

　　TaKaRa Ex Taq(5U/μL) 0.2 μL

　　Ligation Template DNA 10.0 μL

　　ddH2O 4.0 μL

Total Volume 20 μL

　　4. Pipette mix, centrifuge to collect.

　　5. PCR amplification, cycling program as follows:

　　94℃, 5 min

94℃, 30 sec

　　68℃, 30 sec (decrease by 0.7℃ per cycle) 12 Cycles

　　72℃, 1 min

94℃, 30 sec

　　56℃, 30 sec 23 Cycles

　　72℃, 1 min

　　72℃, 5 min

　　4℃ hold.

　　(IV) Polyacrylamide Gel Electrophoresis Detection

　　1. There are various types of commercialized electrophoresis apparatuses, and the configuration between glass plates and spacers also varies slightly depending on the manufacturer, with spacer thickness ranging from 0.5 to 2.0 mm. The thicker the gel, the more heat generated during electrophoresis, and overheating can lead to

　　2. Place the two rubber frames carrying the glass plates into the clamps of the electrophoresis tank, with the short glass side facing inwards. Fix their positions and tighten the screws on both sides and the bottom.

　　3. Take two 50mL small beakers and prepare 40mL of polyacrylamide gel solution for each according to the table below. When preparing the polyacrylamide gel solution, you can first mix water, 40% acrylamide, 5×TBE, and 10% ammonium persulfate, then add TEMED just before pouring the gel, otherwise the gel will polymerize too quickly.

　　4. Lean the electrophoresis tank against the edge of a white ceramic plate, at an angle of approximately 10-20° with the table. Add TEMED to the mixture and swirl the beaker to mix the solution.

Reagents prepared in milliliters for different gel concentrations (%) (Total volume 40mL)

　　6% 5.5% 5% 4%

　　H2O 25.6mL 26.1mL 26.6mL 27.6mL

　　40% Acrylamide 6mL 5.5mL 5mL 4mL

　　5×TBE 8mL 8mL 8mL 8mL

　　10% Ammonium Persulfate 360μL 360μL 360μL 360μL

　　TEMED 40μL 40μL 40μL 40μL

　　5. Pour the solution from the beaker along the side of the long glass plate in the lower rubber frame into the space between the two glass plates, filling it to the top. If there are bubbles, remove them with a thin rod.

　　6. Immediately insert the appropriate comb, with the flat side of the comb close to the long glass plate.

　　7. Invert the electrophoresis tank so that the other rubber frame without gel is at the bottom, and pour the gel using the same method.

　　8. Place the electrophoresis tank upright and allow it to polymerize for 60 minutes at room temperature. If the gel shrinks noticeably, add more acrylamide solution. After complete polymerization, lines with different refractive indices can be seen below the comb.

　　9. If the cooling water circulation device is not connected, use clamps to tighten the two rubber tubes connecting the reservoir.

　　10. After the gel polymerization is complete, carefully remove the comb, immediately rinse the loading wells with water, and fill the buffer tank of the electrophoresis apparatus with 1×TBE.

　　11. Connect the electrodes. The upper reservoir wire is connected to the negative pole of the electrophoresis apparatus (black to black), and the lower reservoir wire is connected to the positive pole of the electrophoresis apparatus (red to red). Set the voltage and current, press the working switch of the electrophoresis apparatus, and perform pre-electrophoresis for 20-30 minutes to remove impurities from the loading wells.

　　12. Place the working switch to the preset position, mix the DNA sample with an appropriate amount of gel loading buffer, use a pipette to draw up the mixture, and inject the sample by positioning the tip close to the long glass plate corresponding to the loading well.

　　13. Set the voltage and current (generally set to constant current 30 mA, or 200V), press the working switch of the electrophoresis apparatus, and perform electrophoresis (approximately 3-4 hours).

　　14. When electrophoresis reaches the position of the standard reference front indicator, cut off the power, unplug the wires, discard the electrophoresis buffer in the buffer tank, unscrew the fixing screws, remove the glass plate and rubber frame, use a thin steel spoon to pry up the upper glass plate from one corner, rinse the gel with a wash bottle so that the gel is completely attached to one glass plate, then rinse the gap between the gel and the glass plate with a wash bottle to completely separate the gel from the glass plate, and use the force of water to rinse the gel into a white porcelain dish (during this process, do not touch the gel with your hands or other tools).

15. Rinse the gel 3 times with deionized water, then place it in fixing solution (the volume of fixing solution should be 0.5cm above the gel surface) for 10 minutes to fix it.

　　16. Pour off the fixing solution, rinse the gel 3 times with deionized water, then place it in staining solution for 10 minutes to stain it.

　　17. Pour off the staining solution, rinse the gel 3 times with deionized water, then place it in developing solution for 10-20 minutes to develop it.

　　18. Once the DNA bands are clearly visible, rinse the gel once more with clean water, then wrap it with cellophane soaked in 10% glycerol (ensure no air bubbles), fix it onto a glass plate with clips, allow it to air dry overnight at room temperature, and after drying, remove the dry gel for band analysis.

　　19. Observation and recording, results analysis.

　　IV. Key Experimental Points

　　1. During enzyme digestion, the general order of adding samples is water, buffer, DNA, and finally enzyme solution.

　　2. Enzyme digestion reaction is a micro-operation. The tip should aspirate from the solution surface to prevent the tip from carrying too much liquid and enzyme. The restriction enzyme to be used should be kept in an ice bath. After use, cap tightly and immediately return to a -20℃ freezer to prevent inactivation of the restriction enzyme.

　　3. Due to temperature differences, moisture often forms on the caps of centrifuge tubes during enzyme digestion reactions. Therefore, after enzyme digestion or when taking samples in between, centrifuge for 2 seconds to concentrate the solution. Otherwise, a decrease in volume will be observed after enzyme digestion.

　　4. If the electrophoresis band is much brighter at the end closer to the loading well than the other end, it indicates incomplete enzyme digestion, which may be due to the following reasons:

　　① Insufficient reaction time (If the reaction is overnight, this point can be excluded).

　　② Insufficient enzyme quantity or partial enzyme inactivation. The latter situation is often encountered when the last tube of enzyme is added.

　　③ If the DNA sample is dissolved in TE buffer and occupies a relatively large volume in the enzymatic reaction, the EDTA in the TE buffer may inhibit the enzymatic reaction. In this case, DNA can be re-precipitated with ethanol. The steps are to add 1/10 volume of 3M NaAC, then add 2.5 times the volume of absolute ethanol, centrifuge (14000g×15min), remove the supernatant, then add 75% ethanol (50 μL) to wash the DNA precipitate, centrifuge again (14000g×15min), remove the supernatant, and after drying, re-dissolve in a small amount of TE buffer or SWD.

　　5. If the DNA bands in the lane are smeared, it may be due to the DNA sample being only partially dissolved in the initial buffer or because ethanol still remains in the DNA sample after ethanol precipitation.

　　6. If the lower part of the electrophoresis band is much brighter than other parts, this may be due to the presence of contaminating RNA or degradation of the DNA sample.

　　V. Required Instruments and Consumables

　　1. 30℃, 37℃, 65℃ Water Baths 2. Electrophoresis Apparatus

　　3. Horizontal Electrophoresis Tank 4. Vertical Plate Electrophoresis Tank

　　5. Pipette 6. Refrigerated Centrifuge

　　7. UV Transilluminator 8. Autoclave

　　9. UV Gel Imaging System 10. Magnetic Stirrer

　　11. pH Meter 12. Shimadzu UV Spectrophotometer

　　13. Ultra-low Temperature Freezer 14. Pure Water System

　　15. Low-temperature Circulating Water Bath 16. Ultracentrifuge

　　17. Refrigerator 18. Forced-air Drying Oven

　　19. Freeze Dryer Centrifuge 20. PCR Machine

　　21. Centrifuge Tubes 22. Various Narrow-mouth Bottles and Beakers

　　23. Clips 24. Cellophane

　　25. Wash Bottles 26. Single-edge Razor Blades

　　27. Pipette Tips 28. PCR Tubes

　　VI. Reagents Required

　　1. Genomic DNA (1mg/mL) 2. Molecular Weight Marker

　　3. 1% Agarose prepared with 1×TBE 4. Ethidium Bromide Stock Solution (10mg/mL)

　　5. Sterilized Distilled Water (SDW) 6. Restriction Endonuclease and Restriction Reaction Buffer

　　7. 3M NaAC 8. Ethanol

　　9. Loading Buffer 10. Tris-HCl

　　11. Boric Acid 12. EDTA (Ethylenediaminetetraacetic acid)

　　13. Ammonium Persulfate 14. TEMED (N,N,N',N'-Tetramethylethylenediamine)

　　15. Glacial Acetic Acid 16. Silver Nitrate

　　17. Sodium Hydroxide 18. Formaldehyde

　　19. Acrylamide 20. N,N'-Methylenebisacrylamide (Bis)

　　VII. Reagent Preparation

　　1. 5×TBE

　　Final Volume 1L Final Volume 500mL

　　Tris-HCl 54g 27g

　　Boric Acid 27.5g 13.75g

　　0.5moL/L EDTA

　　(pH=8.0) 20mL 10mL

　　2.0.5moL/L EDTA(pH=8.0)

　　Add 186.1g EDTA-Na2·2H2O to 800mL water, stir vigorously on a magnetic stirrer, adjust the pH of the solution to 8.0 with NaOH, then make up to 1L, autoclave and aliquot for later use.

　　3.10% Ammonium Persulfate

　　1g ammonium persulfate made up to 10mL or 0.1g ammonium persulfate made up to 1mL.

　　4.TEMED: Commercial Reagent

　　5.Fixing Solution: 10% Ethanol, 0.5% Glacial Acetic Acid

　　Preparation: 50mL ethanol, 2.5mL glacial acetic acid made up to 500mL.

　　6.Staining Solution: 10% Ethanol, 0.5% Glacial Acetic Acid, 0.2% Silver Nitrate

　　Preparation: 50mL ethanol, 2.5mL glacial acetic acid, 1g silver nitrate made up to 500mL, store in the dark.

　　7.Developing Solution: 3% Sodium Hydroxide, 0.5% Formaldehyde

　　Preparation: 15g sodium hydroxide, 6.8 mL 37% formaldehyde made up to 500mL.

　　8.40% Acrylamide (19:1):

　　38g acrylamide, 2g bisacrylamide, add 50mL water, heat the solution to 37°C, make up to 100mL.

　　9.1% Agarose: 0.24g agarose added to 20mL water, heat in a microwave oven.

　　VIII. Discussion Questions

　　1.What are the factors affecting enzyme digestion?

　　2.What are the factors leading to incomplete enzyme digestion reaction?

　　3.Briefly describe the three ligation techniques for sticky ends.

　　4.Compared to agarose gel, what are the main advantages of polyacrylamide gel?

　　5.What is the role of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine (TEMED) in preparing polyacrylamide gel?

　　6.Compare AFLP technique with RAPD technique.

　　Experiment Eight: Bioinformatics

　　I. Related Knowledge

　　(I) Definition of Bioinformatics

　　Bioinformatics is a science that uses computers as tools to store, retrieve, and analyze biological information in life science research. It is one of the major frontier fields in life sciences and natural sciences today, and will also be one of the core fields of natural sciences in the 21st century. Its research focus is mainly reflected in two aspects: genomics and proteomics.

　　Broadly speaking, bioinformatics is engaged in the acquisition, processing, storage, distribution, analysis, and interpretation of biological information related to genome research. This definition includes two levels of meaning: one is the collection, organization, and service of massive data, which means managing these data well; the other is to discover new patterns from them, which means making good use of these data.

　　Specifically, bioinformatics takes the analysis of genomic DNA sequence information as its origin, to find the coding regions representing protein and RNA genes in the genomic sequence; at the same time, to clarify the informational essence of the large number of non-coding regions present in the genome, and to decipher the laws of genetic language hidden in DNA sequences; on this basis, to summarize and organize data related to transcription profiles and protein profiles associated with genomic genetic information release and its regulation, thereby understanding the laws of metabolism, development, differentiation, and evolution.

　　Bioinformatics also uses information from coding regions in the genome to simulate protein spatial structures and predict protein functions, and combines this information with the physiological and biochemical information of organisms and life processes to elucidate their molecular mechanisms, ultimately performing molecular design of proteins and nucleic acids, drug design, and personalized healthcare design.

　　(II) Necessity of Studying Bioinformatics

　　First, with the advent of genome research, there has been an explosive growth in related information, creating an urgent need to process massive amounts of biological information. Since scientists deciphered the 1.8 million nucleotide-long genome of *Haemophilus influenzae* in 1995, approximately 60 microbial genomes and several eukaryotic genomes, such as those of yeast, nematodes, fruit flies, and *Arabidopsis thaliana*, have been completely sequenced to date. By the spring of 2001, scientists had also announced the vast majority of the human genome sequence, i.e., the working draft of the human genome. These achievements mean that genome research will fully enter a new stage of information extraction and data analysis. According to international database statistics, the number of DNA base pairs was 3 billion in December 1999, 6 billion in April 2000, and has now reached 14 billion, roughly doubling every 14 months. At the same time, the growth in digital processing capability of electronic computer chips also roughly doubles every 18 months. Therefore, computers can effectively manage and operate massive data.

　　However, a more fundamental reason is the complexity of genomic data. The genome of a certain organism refers to the sum of all its genetic material. The genetic material of organisms is a type of biomacromolecule called deoxyribonucleic acid (DNA), which is composed of four types of nucleotides linked together, usually represented by the characters A, T, G, C. Popularly speaking, the genetic code of an organism is a linear long chain formed by these four characters linked together. This chain is often very long; for example, the human genetic code contains 3.2 billion characters, which, if stacked, would form an "oracle book" of over a million pages, with 3000 characters per page. This "oracle book" contains vast amounts of information about the structure and function of the human body and life activities, yet it is composed of only four characters, with no morphology, no syntax, and no punctuation, and every page looks similar. How to understand it is an enormous challenge. Genome research ultimately aims to transform biological problems into problems of processing digital symbols. To solve such problems, new analytical theories, methods, techniques, and tools must be developed, and information processing by computers must be relied upon.

　　(III) Current Main Research Content

　　1.Obtain complete genomes of humans and various organisms;

　　2. Discovering new genes and new single nucleotide polymorphisms;

　　3. Non-coding proteins in the genome;

　　4. Studying biological evolution at the genomic level;

　　5. Comparative study of complete genomes;

　　6. From functional genomics to systems biology;

　　7. Protein structure simulation and drug design;

　　8. Research on the application and development of bioinformatics.

　　II. Experimental Principle

　　The main content of this experiment is to compare the expressed sequence tags (ESTs) from sugar beet with information in known databases, thereby mastering the basic methods for functional analysis of unknown sequences.

　　ESTs are gene expression sequence fragments approximately 150-500bp long. EST technology is a method to understand a series of life processes such as growth and development, reproduction and differentiation, genetic variation, and aging and death of organisms, by reverse transcribing mRNA into cDNA, cloning it into a vector to construct a cDNA library, then randomly picking a large number of cDNA clones, performing one-step sequencing of their 5′ or 3′ ends, and comparing the obtained sequences with known sequences in gene databases.

　　Perform homology comparison between EST sequences and GenBank. The Score value indicates the degree of similarity; the higher the Score value, the greater the similarity. The E Value indicates the probability of a random match; the larger the E value, the greater the probability of a random match. Identity indicates the percentage of consistency with the database sequence. Positives indicates the proportion of amino acid properties similar between two sequences. Gaps indicates the proportion of gaps inserted during alignment. Query is the query sequence. Subject is the sequence in the database. When Score <= 80, it indicates that the unknown EST is new. When the Score value is very high, it indicates that the unknown EST has a high degree of homology with the corresponding known gene, from which it can be inferred that this EST has a similar function to the known gene.

　　In EST sequence analysis, the most commonly used method in EST research is sequence similarity comparison to determine the function of ESTs. BLAST (Basic Local Alignment Search ToolA) is one of the most widely used software tools, serving as a package for homology analysis, including 5 software programs: BLASTN, BLASTP, TBLASTN, TBLATX, and BLASTX. BLASTN compares nucleic acid sequences with nucleic acid databases. BLASTP compares amino acid sequences with protein databases. TBLASTN compares protein sequences with all 6 translated sequences in nucleic acid databases. TBLASTX compares all 6 translated sequences of nucleic acid sequences with all 6 translated sequences in nucleic acid databases. BLASTX compares all 6 translated sequences of nucleic acid sequences with protein databases.

　　III. Experimental Methods

　　1. Log in to the National Center for Biotechnology Information (NCBI) website: http://www.ncbi.nlm.nih.gov/. Click BLAST, and a new interface will appear.

　　2. In the new interface that appears, click Nucleotide-nucleotide BLAST (blastn) to enter another interface.

　　3. In the search box on this interface, add the sequence to be analyzed, then click BLAST to search in the non-redundant database nr (non redundant), and a new interface will appear.

　　4. The new interface will display the ID number of the requested sequence. Click Format.

　　5. After waiting for the prompted time, the system will display a series of alignment results for known genes homologous to the analyzed sequence.

　　IV. Required Instruments

　　30 computers (for internet access).

　　V. Discussion Questions

　　1. Definition of bioinformatics?

　　2. Meaning of EST?

　　3. What do BLASTN, BLASTP, TBLASTN, TBLATX, and BLASTX represent in bioinformatics?

　　4. How to perform bioinformatics BLAST analysis on unknown genes or sequences in nr?