18 Molecular Techniques

• Molecular techniques manipulate DNA for analysis, with the length of DNA fragment dependent on differences in repeat units and/or restriction site locations
• Similarities, differences, and relationships between organisms can be identified based on the pattern of separation of DNA fragments
• Principles and procedures of molecular techniques include:

  • Polymerase chain reaction (PCR) with its advantages and limitations
  • Gel electrophoresis
  • Southern blotting and nucleic acid hybridisation
• PCR allows amplification of a specific segment of DNA in vitro, with applications in basic research, human genetics testing, and forensics
• PCR components include:

  • Template DNA
  • Oligonucleotide DNA primers
  • Taq polymerase
  • Deoxyribonucleoside triphosphates (dNTPs)
  • Buffer containing Mg2+
• PCR (Polymerase Chain Reaction) is a technique used to amplify specific segments of DNA
• The basic PCR protocol involves placing all components into a PCR tube and running a standard program in a thermal cycler that heats the tube to different temperatures for different periods of time
• PCR involves three main steps in each cycle:

  1. Denaturation: heat treatment to separate DNA strands

  2. Primer annealing: cooling DNA for primers to attach

  3. Extension: Taq polymerase synthesizes a complementary DNA strand
• Each PCR cycle results in a doubling of DNA molecules being replicated, leading to an exponential increase in the target DNA after multiple cycles
• Advantages of PCR include high sensitivity, speed, specificity, robustness, automation, and ease of use
• Limitations of PCR include Taq polymerase lacking proofreading ability, dependence on accurate DNA sequence information for primers, limited length of DNA fragments that can be amplified, and the risk of contaminating DNA
• PCR applications include clinical diagnosis, pre-natal screening, early detection of infections, paternity testing, forensic analysis, and studying evolutionary relatedness
• Fragments of DNA found in prehistoric specimens can be amplified for further study
• DNA sequences from both living (extant) and extinct organisms can be compared to determine evolutionary relationships
• PCR (Polymerase Chain Reaction) involves two cycles:

  1. In the first cycle, the DNA is denatured, annealed with primers, and extended.

  2. In the second cycle, the DNA is denatured, annealed with primers, and extended
• FAQs to ponder about PCR:

  1. Why is it important for the reaction tube to be thin-walled?

  2. Why is it usually recommended to run about 25 to 30 cycles for PCR?

  3. Why is the discovery of Taq polymerase a breakthrough? What would you do if you have only normal DNA polymerase?

  4. Why is proof-reading activity from 3’ to 5’ and not 5’ to 3’?

  5. Which is worse: an error occurring earlier or later during PCR?

  6. Is it possible to amplify an entire eukaryote or even prokaryotic genome using PCR?
• Gel electrophoresis separates DNA, RNA, or proteins according to molecular size
• Agarose gel electrophoresis principle:

  • Electrophoresis is the movement of charged molecules in an electric field
  • DNA, RNA, and proteins exist as electrically-charged particles at a given pH
  • Agarose gel impedes movement of DNA fragments, affecting longer fragments more than shorter ones
• Agarose gel electrophoresis procedure:

  1. A slab of agarose gel with wells is pre-cast using a comb

  2. The gel is placed in a buffer solution to generate an electric field

  3. DNA samples are mixed with a loading buffer and loaded into wells

  4. DNA ladder, containing DNA fragments of known sizes, is added to one or two lanes

  5. When the current is turned on, DNA fragments migrate towards the positive electrode
• Agarose gel electrophoresis results:

  • DNA fragments separate into bands of different sizes based on their rates of migration
  • Bands are visible after staining with a dye like ethidium bromide and illumination under UV light
  • Gel concentration affects resolution, higher concentration provides better resolution
• When a bacteriophage infects a bacterium, it first injects its DNA into the bacterium
• Cleaving the phage DNA prevents the phage from replicating, conferring resistance to bacteriophage infection to the bacterium
• The bacterium's own DNA is not cleaved because it is methylated at the restriction sites recognized by its own restriction enzyme
• DNA methylation process:

  • A methylase adds methyl groups (–CH3) to adenine or cytosine residues in the DNA sequence that constitutes the restriction site
  • This changes the conformation of the DNA at the restriction site, making it no longer complementary in shape and charge to the enzyme’s active site
  • Methylation protects bacterial DNA from cleavage by its own restriction enzymes
• Restriction enzymes, or restriction endonucleases, break phosphodiester bonds between adjacent nucleotides in DNA
• Most restriction sites are about 4 or 6 bases long and are palindromic, meaning the sequences read from 5’ to 3’ on both strands are the same
• Some restriction enzymes produce sticky ends, resulting from a staggered cut with single-stranded overhangs that can anneal with complementary single-stranded stretches on other DNA molecules cleaved with the same restriction enzyme
• Other restriction enzymes produce blunt ends, made by a simple cut across both strands at a single point
• RFLP (Restriction Fragment Length Polymorphism) is used to detect the sickle-cell anaemia allele by separating DNA fragments through gel electrophoresis to observe different band patterns for genotypes SS, AS, and AA
• Two methods for RFLP detection:

  • Method 1: Extract genomic DNA, digest with MstII, run gel electrophoresis, perform Southern blot with a radioactive probe, and analyze results
  • Method 2: Extract genomic DNA, conduct PCR with primers for the β-globin gene, run gel electrophoresis, and visualize bands under UV light
• RFLP in DNA fingerprinting:

  • No two humans (except identical twins) have the same genome
  • DNA fingerprinting detects varying numbers of tandem repeats, generating a unique DNA fingerprint for each individual
  • Useful in forensic science and paternity testing
• Procedure for RFLP in DNA fingerprinting:

  • Digest with restriction enzymes, separate fragments by gel electrophoresis, perform Southern blotting with radioactive probes, and visualize bands via autoradiography
  • OR design PCR primers, amplify tandem repeat loci via PCR, separate products by gel electrophoresis, and visualize under UV light
• Genetic fingerprinting can be used to verify if a lost animal belonged to the wild or was bred in captivity by comparing genetic fingerprints to conclude the animal's origin
• Explanation of DNA fragment separation in gel electrophoresis:

  • DNA molecules move towards the positively charged electrode in an electric field
  • Agarose gel meshwork impedes longer DNA fragments more than shorter ones, leading to separation based on fragment length