Exercise No. 5 Gender Identification PCR PDF

WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 5 Gender identification based on PCR (Polymerase Chain Reaction) Amplification of the SRY and FMR1 gene sequences Theory Student:  knows the application of PCR  describ...

WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 5 Gender identification based on PCR (Polymerase Chain Reaction) Amplification of the SRY and FMR1 gene sequences Theory Student:  knows the application of PCR  describes and understands the steps of PCR  explains the mechanism of amplification of DNA molecules in PCR  knows the components of PCR and their role  characterizes parameters influencing individual stages of PCR  explains and understands the characteristics of multiplex PCR The PCR (Polymerase Chain Reaction) technique allows for the selective amplification of DNA in vitro by imitating the phenomenon of DNA replication in vivo. It allows for the copying of any given DNA sequence with a length of several hundred to several thousand nucleotides. The PCR method was developed in 1987 by a group of scientists from the Cetus Corporation in the USA. The American biochemist Kary Mullis received a Nobel Prize in chemistry for the development of the foundation of this method. PCR consists of three stages that are repeated between 30 to 40 times: 1. denaturation of the DNA matrix, 2. annealing, hybridization of starters to the complementary sequence of the DNA template, 3. elongation of the primers (synthesis of a new DNA strand). The reaction is conducted in a special apparatus known as the thermal cycler, in which the temperature and the duration of individual stages of the reaction, as well as the number of cycles, are programmed. Ad 1. DENATURATION Above the specified temperature hydrogen bonds between nitric bases of opposite DNA strands maintaining them in the form of a double-stranded DNA helix are too weak and the helix is divided into two strands. In the thermal cycler, for about two minutes, the temperature is raised to approximately 94-95C. In this maximum for Taq polymerase (isolated from the Thermus aquaticus bacteria) temperature, matrix, double-stranded DNA molecules with the content of the G+C bases of up to 55% undergo denaturation and separate into single strands. DNA strands with a higher content of G+C bases require higher temperatures since guanine and thymine are bound by a triple hydrogen bond, while adenine and thymine are bound by a double bond. This is connected with the use of more thermally resistant polymerases such as Pwo and Pfu isolated from the Page 1 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Pyrococcus woesei and Pyrococcus furiosus bacteria respectively. Ad 2 ANNEALING In the second stage, the temperature is lowered to a temperature of 3 -5 C degrees lower than the temperature of melting specified for the starters. At this temperature, the single-strand short DNA segments, the so-called primers, present in the reaction, anneal (attach) within 20-30 seconds to the complementary sequence of the single strand of template DNA. Double-stranded hybrid structures are created at “the end” of each DNA strand meaning at the ends of the DNA fragment that is to be copied. Each of the primers binds only to a single strand of the original DNA. The optimal temperature of primer binding is set out empirically by conducting PCR at a gradient of temperature below melting temperature. There are two types of primers. One of these is referred to as primer 1 or forward primer, the other as primer 2 or reverse primer. Ad 3. ELONGATION In the third stage, the temperature is once again raised to 72C for about 20-60 seconds. At this time Taq polymerase catalyzes the addition of subsequent pairs of bases to the 3’ end of the primer – Page 2 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 complementary to the sequence of the matrix strand, at a speed as high as 2000 nucleotides per minute. In order to estimate elongation time of the copied DNA segment a speed of 1000 nucleotides per second is assumed. Stages I, II, and III are cyclically repeated about 25-35 times. Amplification happens in accordance with the diagram below. In the cycle, the first 2 molecules are created with lengths defined by the location of the starters. In subsequent cycles, the number of multiplied DNA fragments increases exponentially and each new fragment becomes a template in the next cycle. Thermal profile of a PCR cycle cycle 1, cycle 2, denaturation, chain elongation, adding of the starter, time The number of PCR cycles depends on the amount of DNA templates in the reaction mixture as well as the expected efficiency of the PCR product. If the initial number of DNA strands is low, like 10 copies of the DNA, 40 cycles should be performed. If the initial number of DNA strands is higher, then 25-35 cycles is Page 3 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 sufficient. The theoretical effectiveness after “n” cycles equals 2n double-stranded, specific DNA molecules. Exponential growth of DNA copies Cycle Number of DNA chains 1 2 2 4 3 8 4 16 5 32 6 64 20 1.048.576 Page 4 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Amplification diagram of a single DNA strand in the PCR a) schematically represented double DNA strand. The constant line represents the template segment/sequence that is to be copied, the dashed line represents the other DNA strand sequences b) denaturation of the double DNA strand under the influence of a temperature of 95C, breakdown of hydrogen bonds between the strands, and separation of strands into two single strands. Lowering of temperature leads to annealing – the complementary joining of primers/starters (synthetic segments of single DNA strands 5’ →3’forward, 3’ ←5’ reverse) to single DNA template strands. c) Raising of temperature to 72C allows for DNA polymerase to join to the 3’ ends of primers connected to the DNA template and amplification/reconstruction/elongation of the missing complementary strand, a new strand is created d) Newly created strands become the template in the next cycle of DNA amplification e) In the next cycle the primers join to the available, denatured single DNA strands. Then DNA polymerase once again recreates the missing strands leading to another copying of DNA The denaturation process, primer annealing, and their elongation by DNA polymerase is cyclically repeated. In analyzing the creation of new DNA strands we notice that their length is determined by the distance Page 5 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 between the primer forward and primer reverse bond. In the third cycle there are segments created whose length is fully determined by the location of the 5’ →3’forward, 3’ ←5’ reverse primers. PCR components: a. DNA template-sample; single or double DNA strand, linear or circular, b. starters/oligonucleotides/primers with a sequence complementary to the ends of the fragment which is being replicated, c. dNTPs, a mixture of deoxyribonucleotide triphosphates with 200-250 of each of dATP, dTTP, dCTP, and dGTP – "blocks" necessary to create new DNA strands, d. thermally stable DNA polymerase, the most often used is polymerase isolated from Thermus aquatics (Archea) – Taq polymerase, e. type II cations Mg+2- DNA polymerase cofactors. Cations are bonded by DNA and nucleotides (through the phosphorus groups), for each reaction the optimal concentration of Mg+2 is chosen, generally 1.5 mM, f. 10 mM buffer, most often Tris-HCl, pH 8.3 – 8.8, at a temperature of 720C pH approx. 7.2, g. type I cations: 50:100 mM KCl. Optimization of PCR PCR is not usually 100% efficient. In order to increase the efficiency the reaction conditions must be optimally selected, which means they must be altered. Important parameters that allow for the optimization of PCR are the concentration of the DNA matrix, the concentration of the starters, concentration of monovalent and divalent cations, Their appropriate selection (often empirical) increases the specificity of the reaction and the multiplication of the appropriate DNA fragments. A deficiency in the reaction components may lower its efficiency, while their excess may contribute to the creation of non-specific products. The appropriate temperature and the duration of each stage of PCR are also not without significance. It is especially important to select the appropriate annealing temperature i.e. annealing the primers to the DNA matrix since every newly designed pair of primers to replicate a given sequence may have a different optimal annealing temperature. Factors which influence the effectiveness of DNA amplification via the PCR method: 1. annealing and elongation temperature, 2. concentration of the DNA template (amount, purity) and concentration of the starters, 3. MgCl2 concentration, Page 6 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 4. concentration of(dATP, dTTP, dCTP, dGTP), 5. polymerase (Taq) concentration, 6. starter elongation temperature, 7. reaction buffer, reagent concentration, reaction mixture (reaction environment). Ad. 1 The annealing temperature is the most important factor influencing the effectiveness and specificity of PCR. The duration time of this stage depends on the size of the primers (generally it does not exceed 1 minute). The primer annealing temperature should be 5C lower than the calculated starter melting temperature (Tm) If the temperature is too low it causes the starters to anneal at non-specific target sites (based on the principle of incomplete complementarity). The estimated melting temperature ( Tm) of a primer, if the primer is shorter than 25 nucleotides is calculated by using the following formula: Tm= [4 (G + C) + 2 (A + T)] (C) G, C, A, T – corresponds to the amount of individual nucleotides in a primer. If the primer is longer than 25 nucleotides, melting temperature ( Tm) should be determined by using specialized computer programs, which take into account the mutual influence of neighboring bases, influence of salt concentration, etc. Ad 2. The concentration of matrix DNA should fall between:  0,01-1 ng for plasmid or phage DNA,  0,1-1 µg for genome DNA. A higher concentration of DNA increases the content of non-specific PCR products. The concentration of each of the primers of the reaction should be between 0.05 - 1 μM, and optimally between 0.2 - 0.3 μM. Too low primer concentration limits the effectiveness of the reaction, while too high concentration causes non-specific PCR products to appear. Ad. 3 All polymerases require the presence of type II cations to work, and these are generally magnesium ions (Mg+2). the amount of Mg+2 molecules must exceed the amount of phosphorus groups present in the reaction Page 7 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 mixture, since both free nucleotides, as well as the starters, bind Mg+2 ions. The final Mg+2 concentration generally equals between 0.5 to 5 mM. The optimal concentration for most polymerases is between 1.5-2.0 mM. Some polymerases also require manganese ions (Mn+2). Ad. 4 The used dNTP mix should contain equal amounts of four nucleotides dATP, dTTP, dCTP, and dGTP. An uneven amount of dNTP reduces amplification efficiency. The optimal dNTP concentration should equal between 200 to 400 μM. Too high a concentration of dNTP may bind Mg2+ ions decreasing their availability for polymerase. A low concentration of dNTP increases the accuracy of DNA replication, however, it decreases the efficiency of this process. Ad 5 Taq polymerase was obtained from the Thermus aquaticus bacteria or Escherichia coli recombinants. A characteristic property of this enzyme is thermal stability within a range of temperatures between 37-94C. Taq polymerase activities are:  the ability to synthesize DNA strands from the 5’ end to the 3’ end which requires the presence of a single-strand template and DNA primers,  the ability to degrade single-stand or double-strand DNA meaning the activation of 5’-3’ exonuclease,  a lack of ability to cut out a wrongly inserted nucleotide (the amount of wrongly inserted nucleotides equals 2 x 10-4 do 2 x 10-5), meaning it does not possess the activity of 3’-5 exonuclease. The concentration of Taq polymerase should equal 1U per reaction 0.04-0.1 U/μl ). Higher concentrations of polymerase may cause the synthesis of non-specific products. If there are various kinds of inhibitors present (e.g. if the DNA matrix used is not thoroughly purified) in the reaction mixture, then higher concentrations of polymerase are recommended. Ad 6 Temperatures of elongation should fall within the range of 65 to 75C, but most often it is 72C. A lowering of the elongation temperature (60-68C) which results in extended duration time is beneficial if:  greater accuracy is required in the synthesis of a new strand, e.g., in the reaction of cyclical sequencing,  the amplified fragment will be cloned into expression vectors – genetic engineering The elongation speed, depending on the type of polymerase is between 1kb/min to several kb/min. Generally Page 8 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 in planning the duration of the elongation stage of PCR, it is assumed that polymerase speed equals 1kb/min. Ad 7 The PCR reaction buffer is specific for a given polymerase and is generally supplied along with the polymerase. It stabilizes the pH of the reaction mixture. The buffer contains type I potassium chloride (KCl) cations. The standard concentration of KCl equals approx.. 50mM and it enables the amplification of fragments over 500bp. Higher KCl concentration (70-100 mM) improves the amplification of fragments over 500 bp. The reaction mixture apart from the Tris buffer with a pH of 8, often contains other additives such as:  bovine serum albumin (BSA),  ammonium sulfate (NH4)2SO4,  Trition chemical detergent. These additives may stabilize the polymerase and modify the reactions between the matrix and the starters. Factors that decrease the effectiveness of PCR:  proteinase K used in cell lysis during DNA isolation may degrade the DNA proteolytically,  detergents, especially ion detergents,  the presence of phenol inhibits the activity of DNA polymerase,  sodium concentration (e.g. ammonium chloride) in the reaction mixture influences the activity of Taq polymerase,  potassium ions influence the Tm of primers,  EDTA chelates divalent ions, meaning it eliminates them. Thus divalent ions as the necessary cofactors of DNA polymerase become unavailable. Page 9 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Factors improving PCR effectiveness: Certain compounds added to the reaction mixture may (in a limited range of concentrations) improve the effectiveness of PCR. Adding DMSO (dimethyl-sulfoxide):  may eliminate the creation of secondary structures by primers,  lowers melting temperature ( Tm) by 5-6C. Adding glycerol causes:  improved denaturation of the DNA template,  elimination of the creation of secondary structures of primers and the DNA template. Threats to PCR False positive results:  contamination with post-amplification product,  contamination of reagents,  contamination of the examined samples with the control DNA matrix while preparing the positive control. False negative results may be caused by:  reaction inhibitors,  an error in tube volume,  a mistake made by the person doing the exercise,  low quality of the isolated nucleic acids  problems with the equipment (e.g. thermal cycler).  problems connected with the reagents (magnesium concentration, primer synthesis). Page 10 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 5 Gender identification based on PCR (Polymerase Chain Reaction) Amplification of the SRY and FMR1 gene sequences Practice Student:  knows the practical rules of performing a PCR in a laboratory,  can plan and design a PCR taking into account the number of examined DNA samples and their concentrations,  understands and explains the use of the appropriate control reactions. Performing a PCR (Multiplex PCR) In the practical part of the exercise, a PCR will be performed on a previously isolated genome DNA from cells collected from cheeks to identify the presence of the sequence of an SRY gene located only on the Y chromosome. The SRY gene plays a key role in the shaping of the biological gender of a person. A lack of the SRY gene in the male embryo will lead to the development of female phenotype characteristics and a lack of fertility.  the presence of a PCR product amplified with specific starters of the SRY genes confirms the male gender.  lack of the product means a lack of the presence of the SRY gene in the examined sample of genome DNA and therefore female gender. However, attention must be paid to the fact that a lack of PCR product as a false negative result of a PCR reaction may be caused by the lack of occurrence of a PCR due to technical reasons e.g.,  inappropriate composition of the reaction mixture,  bad quality of the genome DNA sample. That is why to confirm the accuracy of a negative result, meaning a lack of specific product for the SRY gene sequence an internal control PCR is conducted, e.g., with primers specific for the FMR1 gene found on the X chromosome. The presence of a specific product of the FMR1 gene is expected both for the male, as well as the female genetic material – it confirms the appropriate course of the PCR reaction. The characteristics of the SRY gene find applications in molecular diagnosis as a marker:  in determining biological gender,  in the case of pathologically developed male or female gonads,  in the non-invasive determination of the fetal gender,  in criminalistics in determining relationships. Page 11 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 In the present exercise the conducted Multiplex PCR contains two pairs of primers for two separate gene sequences; target SRY and control FMR1 and thus it is a variation of the basic reaction which uses one pair of primers. Multiplex PCR allows for simultaneous identification of several DNA sequences in a single sample of material under the condition that the designed primers replicate sequences of various lengths of nucleotides providing products that may be separated and identified by conducting DNA electrophoresis. Multiplex PCR requires a more advanced optimization of the selection of starter sequences in order to limit the self hybridization of primers and the amplification of non-specific DNA fragments. !To perform the exercise only reagents designated for scientific purposes and not for diagnostic will be used. The obtained results, therefore, do not have diagnostic capabilities, and are only used to complete the present exercise and become familiar with the methodology of PCR! Preparation of PCR Equipment: automatic pipettes, pipette endings, vortex, microcentrifuge, Eppendorf tubes, eight 0.2 ml tubes for PCR, thermal cycler. Reagents: “PCR Mix Plus Green”, water free of nucleases, primers, DNA samples (matrix) For the purpose of the present exercise commercially available reaction mixture “PCR Mix Plus Green” will be used. It is a ready and optimized mixture when it comes to the concentrations of single and universal components needed for a standard PCR.. Alternatively, the PCR may also be prepared from single components by appropriately selecting and optimizing their concentrations. „PCR Mix Plus Green” contains:  Taq DNA polymerase (0,1 U/µl),  MgCl2 (4 mM),  dNTPs - 0,5 mM of each of the deoxynucleotides (dATP, dCTP, dGTP, dTTP). Apart from the above components necessary in the PCR, the “PCR Mix Plus Green” contains a blue and yellow dye and a loading buffer. The presence of dyes and the loading buffer enables the application of the examined reaction onto the electrophoretic gel directly after the PCR reaction is completed in the thermal cycler. The loading buffer prevents the sample from flowing out of the well in the agarose gel, while the dye possessing electrophoretic properties allows for monitoring of the progress of the electrophoresis itself Page 12 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 during its course. These dyes, do not yet enable the visualization of the DNA replicated in the PCR. Protocol of preparing PCR with the use of a ready-made reaction mixture 1) „PCR Mix Plus Green”, the water free of nucleases, and the isolated DNA samples must be defrosted on ice and carefully mixed by reversing the tubes. Then the tubes must be briefly centrifuged and put into the ice (After use “PCR Mix Plus Green” must be stored at -20C). Cyclical seven-times defrosting and freezing do not influence the efficiency and activity of the product. 2) „PCR Mix Plus Green” is double concentrated. Adding appropriate volume of the remaining reaction components to "PCR Mix Plus Green", i.e. starters, DNA matrix, as well as water, dilutes the "PCR Mix Plus Green" to a single/working concentration. This working concentration of the mix ensures the optimal concentration of the individual components of the reaction. 3) While defrosting the reagents each two-person team based on the concentration of the obtained DNA sample calculates the volume which must be used to add to the PCR 100 ng of the DNA sample. Adding a constant amount of the examined DNA samples to the PCR e.g., 1000 ng eliminates the influence of DNA concentration in PCR on its efficiency. Example: if the concentration of the DNA sample equals18 ng/µl, then 8,33ul DNA is collected:  18 ng – 1 ul,  100 ng – X X = 5,55ul DNA Comment: To limit the risk of errors while pipetting the components for each PCR separately, the so- called common MasterMix for N number of PCR reactions is used. Prior to the preparation of the MasterMix, we must answer the question of how many samples must be examined and which control reactions should be taken into account. Ultimately, to prepare the appropriate volume of the MasterMix we need the total number of PCRs (amount of samples + amount of control reactions) increased by 2 or 3 additional reactions. The additional reactions make up for the losses in MasterMix volume resulting from pipetting and portioning of the MasterMix for single reactions. Page 13 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 4) Sample preparation diagram: I M K A B C P1 (N) Subgroup I (6 people, left side) Sample ID sample conc. vol. of DNA vol. of H2O ng/µl µl µl 1) positive male control (M), 2) positive female control (F), 3) tested DNA sample (A), 4) tested DNA sample (B), 5) tested DNA sample (C), 6) tested DNA sample (P2 indicated by the teacher), 7) negative control (N). II M K D E F P2 (N) Subgroup II (6 people, left side) Sample ID sample conc. vol. of DNA vol. of H2O ng/µl µl µl 1) positive male control (M), 2) positive female control (F), 3) tested DNA sample (D), 4) tested DNA sample (E), 5) tested DNA sample (F), 6) tested DNA sample (P2 indicated by the teacher), 7) negative control (N). Page 14 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Total PCR reactions that must be prepared for both groups:…………… Planned surplus reactions:…………….. Total number of reactions that must be prepared to conduct the PCR test:…N=………. Comment: A positive control is a sample that we know contains the examined DNA sequence, while the quality of this DNA material allows for the replication of this sequence via the PCR method. (Generally, this is the sample of the tested DNA, which in previously conducted PCRs yielded the expected results – meaning the amplified product). In such a control PCR reaction it is assumed that the expected PCR amplification products will be obtained. A lack of the product means an inappropriate PCR reaction (please provide possible reasons). ………………………………………………………………........................................................................ ……………………………………………………………………………………………………………… When the PCR product appears neither in the positive control nor in the tested samples, the result of the PCR cannot be properly interpreted – and it cannot clearly confirm that the examined sample does not contain the sequence in question. The obtained results can therefore be false negative. The negative control, meaning the one containing H2O instead of the DNA template. Despite all the necessary components, PCR does not take place. The expected result of the reaction is a lack of the PCR product, such a control result confirms the purity of the individual components in the MasterMix and allows for the appropriate interpretation of the tested samples. The presence of the PCR product in this control means contamination of the reaction components with DNA material of unknown origin. Likewise, the appearance of the product in the other tested samples may be connected with contamination of the PCR, and not with the actual presence of the tested sequence in the examined sample – the result obtained is false positive. Page 15 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 5) In a 1.5ml tube (free of DNases) one MasterMix for both subgroups will be prepared in accordance with the table found below. The MasterMix contains multiple volumes of each of the components of a single PCR without the DNA matrix. Component concentration Volumes of Concentrati MasterMix per N……. reactions components per 1 on in in 25µl reaction in 25 µl reaction PCR Mix Plus Green 2x 12.5 µl 1x ……… µl Starter SRY F (10 µM) 0.5 µl 0,2 µM ………µl Starter SRY R (10 µM) 0.5 µl 0,2 µM ………µl Starter FMR1 F (10 µM) 0.5 µl 0,2 µM ………µl Starter FMR1 R (10 µM) 0.5 µl 0,2 µM ………µl Water free of nucleases 0.5 µl - ………µl ************************ *********** ******* ************************** Total volumes w/o the matrix 15 µl ………µl DNA matrix 100 ng X µl 4 ng/µl Matrix is added to the MasterMix Water free of nucleases 10µl - X µl separately after its portioning into 15 µl Final total of reaction volumes 25 µl --- Page 16 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 6) In accordance with the diagram below after portioning MasterMix into single tubes, each team adds 100ng of DNA of the obtained sample. The DNA volume X µl was calculated at point no. 3. Then each individual reaction has to be topped off with water free of nucleases to reach the final reaction volume of 25µl. The volume of the added water is a result of the difference in the target volume of the reaction 25µl and the amount of the already added components (15 µl MasterMix + X µl DNA). MasterMix portioned into 15 μl, Adding 100 ng (X μl) of the DNA template of individual DNA samples and the appropriate controls. Topping off the reaction to reach 25 μl with water free of nucleases (10 μl-X μl) 7) The closed tubes should be stored on ice until the thermal cycler program is prepared, 8) Programming the thermal cycler in accordance with the sequence below: a) initial DNA denaturation 94C - 2 minutes, b) cycle 1 I) DNA denaturation in the cycle 94C - 15 seconds, II) starter annealing 57C - 20 seconds, III) starter elongation 72C - 20 seconds, c) repetition of b) to repeat the amplification cycle - setting 34 cycles d) final elongation 72C - 10 minutes, e) finishing the reaction 4C 9) The samples must be frozen at -20C until the next exercise, during which DNA electrophoresis will be conducted allowing for the evaluation of the products of PCR as far as their presence and mass. Page 17 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Primer sequence table Primers Primer sequence Primer SRY F (10 µM) 5’-CAT GAA CGC ATT CAT CGT GTG GTC-3’ Primer SRY R (10 µM) 5’-CTG CGG GAA GCA AAC TGC AAT TCT T-3’ Primer FMR1 F (10 µM) 5’-CCC TGA TGA AGA ACT TGT ATC TC-3’ Primer FMR2 R(10 µM) 5’-GAA ATT ACA CAC ATA GGT GGC ACT-3’ Size of the expected PCR products SRY F and SRY R primers give products with a length of 254 bp. FMR1 F and FMR1 R primers give products with a length of 301 bp. Fragments of gene sequence for which starters were designed in the present exercise Product PCR 254 pz Page 18 z 19  WORKSHEET 5- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Product PCR 301 pz Preparation for exercises: Theory regarding PCR (from exercise no. 3 – PCR presentation, and the present instruction no. 5) Required literature: “Essential Cell Biology” B. Alberts Chapter 6, Recommended additional literature: “Gene Cloning: principles and applications” Julia Lodge, ISBN: 0-7487-6534-4 “Rapid sex determination using PCR technique compared to classic cytogenetics”, Settin A1, Elsobky E, Hammad A, Al-Erany A. Int J Health Sci (Qassim). 2008 Jan;2(1):49-52. Page 19 z 19