YIJC [H2] CI2.9 Molecular Techniques (N)(S) (wo checkpoint answers)

2024 JC1 BIOLOGY LECTURE NOTES CORE IDEA 2: GENETICS AND INHERITANCE TOPIC 2.9: DNA MOLECULAR TECHNIQUES Learning Outcomes: (k) Describe the principles and procedures of these molecular techniques: i. polymerase chain reaction (including advantages and limitations) ii. gel electrophoresis iii. Southern blotting and nucleic acid hybridisation References: Reece, J. B., et al. (2011). Campbell Biology (9th Ed), Chapter 20: Biotechnology, pp449 – 453 1 INTRODUCTION  Gene expression can be studied using fundamental techniques of molecular biology such as the polymerase chain reaction (PCR), gel electrophoresis, Southern blotting and nucleic acid hybridisation.  Such molecular techniques can separate and visualise specific fragments of DNA that are of interest e.g. disease-causing alleles in sickle cell anemia. 2 POLYMERASE CHAIN REACTION The Polymerase Chain Reaction (PCR) can rapidly amplify a specific segment of DNA in vitro (outside the cell) for the purpose of cloning and analysis. The method is designed to permit selective amplification of a specific target DNA sequence within a heterogenous collection of DNA sequence. Possible applications of PCR: - Amplify specific gene or DNA fragment - DNA fingerprinting - Studying human ancestry - Paternity testing Learning Outcome 2(k)(i): Describe the principles and procedure of polymerase chain reaction (including advantages and limitations). H2

2 2.1 Reagents (i) Target DNA Sequence Target sequence refers to DNA to be copied, usually a gene of interest which is to be studied. The target sequence to be amplified is usually ~1 to 2 kb in length. (ii) Primers Primers are synthetic short DNA sequences (single -stranded oligonucleotides) approximately 20 – 30 bp in length. Primers (forward and reverse primers) are complementary to DNA sequences at the end of the DNA target sequence. o The forward primer will hybridise to the DNA coding stranding, 3’ to 5’ direction o The reverse primer will hybridise to the DNA non coding strand, 5’ to 3’ direction o The primer provides a free 3’ OH end for DNA polymerase to replicate the target sequence by elongating the DNA strand in the 5’ to 3’ direction. This allows the replication of the double-stranded DNA molecule. Target DNA sequence Fig. 2.1.1: Use of primer to initiate PCR process CHECKPOINT 1 Deduce the sequence of two 5-nucleotide long primers that can be used to amplify the complete target DNA segment. Specify the polarity of your primers. target DNA sequence 5’ T T C G A T G C C T A T G C T A T G C C 3’ 3’ A A G C T A C G G A T A C G A T A C G G 5’ primer 1 5’ G G C A T 3’ primer 2 5’ T T C G A 3’ *Recall, DNA can only be synthesized in the 5’ to 3’ direction *Common misconception by students: The primers are complementary to each other. The example clearly shows that the primers are not complementary. The 1st primer (forward primer) hybridise with coding strand (3’  5’), Allow for replication of coding strand A 2nd primer (reverse primer) will hybridise with non-coding strand (5’  3’), allow for replication of non-coding strand

3 (iii) DNA Polymerase A DNA polymerase derived from a thermophilic bacterium, Thermus aquaticus (Taq polymerase) is used in PCR. Taq polymerase is thermally stable (i.e. capable of withstanding high temperatures), with an optimum temperature of ~75C. This is important as the PCR process requires repeated cycles of heating and cooling. Taq polymerase binds to the 3’ OH end of the primer, adding deoxyribonucleotides complementary to the target sequence in a 5’  3’ direction, to elongate the DNA strand. (iv) Free Deoxyribonucleotides (dNTP) The four types of deoxyribonucleotides, dATP, dTTP, dGTP and dGTP, are added in approximately equal proportions in excess. 2.2 Process  The process involves a 3-stage cycle of denaturation, annealing and elongation which results in an exponential increase in the number of copies of the target DNA molecule of identical sequences.  PCR reagents are added in a reaction tube and PCR is carried out in a thermal reactor, known as a thermal cycler , which automates the temperature and timing of each step in the amplification cycle. Stage 1: DNA denaturation The target DNA sample and required reagents are heated to ~ 95°C for 1 min, to denature and separate the double-stranded (ds) DNA strands into single-stranded (ss) DNA. Heating increases kinetic energy of DNA molecules, resulting in breaking of hydrogen bonds between complementary base pairs. Stage 2: Annealing The mixture is then cooled to ~ 55°C for 2 min, in the presence of excess primers. Forward and reverse primers would bind to complementary sequences flanking the target DNA sequence via hydrogen bonds. Due to presence of excess primers, the probability of primer annealing is higher than the DNA target sequence renaturing. Denaturation @ 95oC dsDNA denature & separate into 2 ssDNA. Annealing @ 55oC Excess primers anneal to complementary base pair at sequence flanking target DNA.

4 Stage 3: Elongation The temperature is raised to 72°C for 3 min. Taq polymerase synthesizes the complementary DNA strand in the 5’ to 3’ direction by adding free dNTPs to the free 3' OH end of the primers using the target DNA sequence acts as template.  After the first cycle, the same process (denaturation, annealing, elongation) is repeated 20 to 30 times in an automated thermal cycler. Fig. 2.2.2: PCR process Elongation @ 72oC Taq polymerase extends forward & reverse primers at free 3' OH ends. Fig. 2.2.1: One PCR cycle

5  Each cycle results in the doubling of the target DNA sequence. Thus, number of copies of dsDNA is 2n where, n is the number of cycles. Fig. 2.2.3: Exponential increase in DNA copies CHECKPOINT 2 Calculate the number of DNA molecules (double-stranded) and DNA strands (single-stranded) after each PCR cycle. Cycle Number of target DNA molecules (ds) Number of DNA strands (ss) 0 1 2 1 2 3 30 n

6 CHECKPOINT 3 (a) Label the graph to show the stages of polymerase chain reaction. (b) Label the appropriate temperature on the y-axis and time on the x-axis. time / min temperature / oC

7 2.3 Advantages  Sensitive The process is sensitive and can amplify minute samples of target DNA. In criminal investigations, DNA fingerprints can thus be prepared from cells in a tiny speck of blood or from the base of a single human hair. In pediatric medicine, physicians can detect genetic defects in very early embryos by collecting a few cells and amplifying their DNA.  Robust PCR is a robust process which can amplify DNA sequences from badly degraded material or even DNA embedded in material that is difficult to extract, as long as a few molecules contain the complete target sequence.  Rapid and relatively easy to use Each cycle takes only 3 - 5 min. Thus, a PCR reaction involving 30 cycles of denaturation, annealing and elongation can be completed in a few hours , yielding a large number of DNA molecules and amplifying the target sequence. PCR can be performed using relatively simple equipment, a thermal cycler. Reagents are added to reaction tubes in appropriate amounts and conditions (e.g temperature, duration, number of cycles) are set. The reaction process is then fully automated and the cycles can run unattended.  Relatively high fidelity The amplification is relatively accurate with error rates ranging between 1 in 10,000 bases to 1 in 100,000 bases. Error rates vary with the choice of polymerase. 2.4 Limitations  Too sensitive Even a tiny amount of contaminant DNA in a sample may become amplified if it includes a DNA sequence complementary to the primers, potentially leading to a e