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

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