MOLECULAR METHODS: GENOTYPIC IDENTIFICATION

• TARGET MICROORGANISMS FOR MOLECULAR-BASED TESTING

  • Those that are difficult or time-consuming to isolate.
  • e.g., Mycobacteria (grows as long as 40 days)
  • Hazardous organisms 
  • e.g., Histoplasma, Coccidioides
  • Those without reliable testing methods
  • e.g., Mycoplasma (common contaminant of cultures)
  • High-volume tests
  • e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis
• USING GENOTYPIC IDENTIFICATION

  • Detecting specific nucleotide sequences
  • These tests can identify sequences unique to species or groups.
  • Nucleic acid probes
  • Nucleic acid amplification tests (NAATs)
  • Limitation is that each detects only a single possibility, not multiple at the same time.
  • It is needed to run multiple probes if the organism being tested could be one of the multiple different species or related groups but it has a maximum number.
• USING GENOTYPIC IDENTIFICATION

  • The nucleic acid probes locate the nucleotide sequence characteristic of species or group.
  • Most methods first increase the DNA in the sample to amplify it.
  • e.g., inoculation on agar or in vitro DNA amplification
• USING GENOTYPIC IDENTIFICATION

  • The nucleic acid probes locate the nucleotide sequence characteristic of species or group.
  • Fluorescence in situ hybridization (FISH) probes for 16S rRNA
  • 1.A double-stranded DNA sequence that is unique to target organism X.
  • 2. The DNA is labeled, then denatured to become a probe.
  • 3. Next, a probe is added to the denatured, single-stranded DNA of the unknown organism.
  • 4. Once it opens up, two results may occur:

  1. If the probe does not bind to DNA, then unknown organism is not organism X.
  2. If the probe binds to DNA, then unknown organism is organism X.
• • A limiting factor is that there should be an idea on the organism first as there is no way to design a probe of an unknown organism. Although, there are random probes that are being commercially used which is the most common way right now, but it is based on sequencing as well. One has to do sequencing later on and a reason on why assays are being used mostly for research purposes only.
• • Nucleic acid amplification tests (NAATs) are used to increase the number of copies of specific DNA sequences especially in low numbers.
  • It allows the detection of small numbers of organisms.
  • Often from body fluids, soil, food, and water as it takes time for them to grow in culture media because the starting amount is very small, too few to start and usually dies.
• Nucleic acid amplification tests (NAATs)

  • It is used primarily for the detection of organisms that cannot be cultured, an unknown organism that has not been cultured at all or not yet been identified.
  • Polymerase chain reaction (PCR) is a common NAAT technique.
• NAATs: PCR SEQUENCING AND DNA HYBRIDIZATION

  • Using molecular techniques such as PCR to examine DNA sequences can help to identify what strain of a pathogen is present in a specimen.
  • Polymerase chain reaction or PCR is a technique that makes multiple copies of a piece of DNA or RNA in a process called amplification.
  • Amplification makes it easier to detect the tiny strands of an organism’s DNA.
• NAATs: PCR SEQUENCING AND DNA HYBRIDIZATION

  • PCR can start with very small amounts of DNA and can be used with viruses or bacteria.
• • The steps in PCR are the following:

  1. PCR starts with a sample of DNA from a clinical specimen suspected to contain a pathogen.

  • 2. A primer, similar to a probe, is added to the sample.
  • 3 Other materials are added to the mixture which include a polymerase enzyme and the “building blocks” of DNA bases
• • PCR consists of three steps, going around for 30 to 40 cycles:

  1. Denaturation

  • Heat for 1 minute at 94°C which causes the strands to split.

  1. Annealing

  • Heat for 45 seconds at 54°C which causes the complimentary forward and reverse primers to start to fall to each other.

  1. Extension

  • Heat for 2 minutes at 72°C which causes the dNTPs or the “building blocks” to be used to fill out the remaining strands. dNTP stands for deoxynucleotide triphosphate.
• PCR

  • One example is if it is believed that Salmonella is causing an outbreak of diarrheal illness, a gene that is unique to Salmonella should be amplified, which makes it a challenge. After the PCR reaction, the amplified genes by PCR would be used to confirm if the organism is Salmonella. It is important to ensure that proper collection, shipment, and storage of the sample have taken place in order to be successful.
• 16S rRNA Sequence Analysis

  • Sequencing RIbosomal RNA Genes
  • Ribosomal RNA (rRNAs) or encoding DNA (rDNAs)
  • Sequences relatively stable
  • Ribosomes would not function if there are too many mutations.
  • 16s rRNA most useful because of moderate size
  • Only around ~1500 nucleotides 
  • PCR can only amplify up to around 2000 nucleotides accurately.
• 16S rRNA Sequence Analysis

  • 16S (18S in eukaryotes) RNAs are small subunit (SS or SSU) rRNAs
  • Sequence compared with extensive databases
  • Can be used to identify organisms that cannot be grown in culture
• 16S rDNA Sequence Analysis

  • Comparisons revolutionized classification
  • Sequences highly conserved since function is critical
  • Lack of mutations allows identification of distant relatedness
  • Certain regions are relatively variable, allowing the determination of recent divergence (if there is a new mutation, it can be a point of recent divergence).
  • Horizontal gene transfer appears rare.
  • Culturing not necessary
  • May not resolve at species level since closely related prokaryotes can have identical 16S rDNA sequences
  • In such cases, DNA hybridization is a better tool
• DNA Hybridization

  • Relatedness of organisms can be determined by similarity of nucleotide sequences
  • Sequence homology measured
  • Extent of hybridization reflects degree of similarity
  • Complementary base pairing of single strands are used
• DNA Hybridization

  • If high percentage, considered related
  • Rule of thumb: 70% similarity is often considered as the same species.
  • Depends on the researcher; cut-off is arbitrary
  • However, it does not always accurately describe relatedness (e.g. Shigella and Escherichia should be grouped in the same species based on DNA hybridization.
• Sample Case

  We can see that both organisms are similar to each other except for a few nucleotides.

  • Shows us the arbitrary limitations of sequencing because the conventions change from one time to another. This is the reason why taxonomic classifications get updated
  • Also applicable to viruses, which change more frequently than bacteria.

  
• DNA sequencing is the process of working out the order of the building blocks or bases, in a strand of DNA. These are the steps:

  1. Before sequencing, it has to be cut up into smaller pieces that are inserted into plasmid DNA and then put into bacterial cells, making it possible to produce lots of copies as the bacteria cells multiply.
  2. DNA is then isolated from the bacteria and sent for sequencing.
  3. The isolated DNA is transferred to a plate where the sequencing reaction will take place.
• DNA Sequencing - 3D (Video)

  • 4. A mixture of ingredients is added which includes free DNA bases (A, C, G, T), DNA polymerase enzyme and DNA primers. Also, terminator bases, modified DNA bases labeled with colored fluorescent tags, are added.
  • 5. To start the reaction, everything is heated to 96°C which separates the DNA into two single strands.
  • 6. The temperature is then lowered to 50°C as this enables the DNA primers to bind to the plasmid DNA.
• DNA Sequencing - 3D (Video)

  • 7. It is then increased to 60°C and the enzyme DNA polymerase binds to the primer DNA, starting to make a new strand of DNA by adding unlabeled DNA bases to the target DNA. It continues to add DNA bases until a terminator base is added. These terminator bases have been chemically altered so that no more bases can be added to the new strand of DNA.
• DNA Sequencing - 3D (Video)

  • 8. To separate the new DNA strand from the original strand, it is heated to 96°C again. This process of heating and cooling is repeated again and again to produce lots of fragments of DNA of different lengths.
• DNA Sequencing - 3D (Video)

  • 9. Various fragments are separated by length using electrophoresis. A capillary tube is lowered into each well of the plate and an electrical charge is applied. This causes the negatively charged DNA molecules to move through the capillary tube. Each capillary contains a porous gel. The shorter fragments of DNA moved through the gel more easily than the longer DNA fragments. As a result, the fragments become arranged by size from the shortest to the longest.
• DNA Sequencing - 3D (Video)

  • 10 Thus, as the DNA fragments come to the end of the capillary, a laser makes the terminator bases light up and the color is detected by a camera and recorded. Each terminator base is labeled with a different color: A (fluorescent green), C (blue), G (yellow), and T (red). The shortest DNA fragments will be read first and the longest read last
• DNA Sequencing - 3D (Video)

  • 11 The sequencing machine records the color of the terminator bases as a series of colored blocks. Each colored block represents the labeled terminator base at the end of each fragment of DNA. By converting the colors into letters, the sequence of the piece of DNA is attained.
• Sequencing at Speed (Video)

  This process is called massively parallel sequencing because millions of clusters are sequenced and imaged simultaneously.

  1. Preparing the DNA: it is broken into fragments that are 200-600 base pairs long. Adenine base is added to the 3’ of each fragment. Adapters, short sequences of DNA, are attached to the ends and then the double strands of DNA are separated into single strands
• Sequencing at Speed (Video)

  • 2.Flow Cell: The single strands are washed across a flow cell, a small plastic slide with lots of small pieces of DNA called primers. The DNA will bind to the complementary primers on the flow cell and then, any DNA that does not attach will be washed away.
• Sequencing at Speed (Video)

  • 3. Flow Cell: DNA bases and DNA polymerase enzymes are added so that the complementary strand of DNA is made. The attached DNA fragments are used as templates to make many copies. The DNA strands bend over and attach to the primers in the surface of the flow cell to form a bridge. The complementary strand is made along the bridge and is repeated many times. The DNA is denatured leaving clusters of single stranded DNA. 
• Sequencing at Speed (Video)

  • 4.. Sequencing: Primers, polymerase enzymes, and fluorescently labeled DNA bases are added. The primers are attached to the sequenced DNA. The polymerase enzyme binds to the DNA and adds a complementary fluorescently labeled DNA base. These bases are reversible terminator bases which means other bases cannot be added after. 
• Sequencing at Speed (Video)

  • 5. Sequencing: Each of the terminator bases give off a different color. Lasers pass over the flow cell causing the bases to glow. The glow is detected by a digital camera. Once it is detected, the fluorescent terminator tag is removed from the base to allow a new terminator base to be added. This process is repeated over and over until about 120 DNA bases have been sequenced. 
• Sequencing at Speed (Video)

  • 6. Analysis: The DNA sequence of each cluster is exported from the sequencer machine for analysis. 
• DNA Hybridization (Video)

  • It involves the formation of a double-stranded nucleic acid and involves the use of a single-stranded radioactive probe sequence from a cloned gene. This is the basis for various DNA probe techniques. 
  • Relatedness of organisms can be determined by similarity of nucleotide sequences. 70% similarity is often considered the same species.
• DNA Hybridization (Video)

  1. At high temperatures: Double stranded DNA will denature or separate into single strands.
  2. When temperature goes lower: The two strands will anneal because of the base pairing interaction of the complementary strands.
• DNA Hybridization (Video)

  3. Hybridization: If the nucleotide sequences are similar, complementary strands from different sources will also anneal to indicate that each strand comes from a different source. Regions of double stranded DNA in which the respective single strands hybridized to one another are said to be homologous (same nucleotide sequences).
• DNA Hybridization (Video)

  • 4. The high specificity of base pairing interactions between complementary strands of DNA can be used to locate a specific nucleotide sequence in a sample. If the DNA from one source is immobilized by attachment to a solid surface such as nitrocellulose, homologous DNA from another source will hybridize and be retained by the immobilized DNA. Non homologous DNA will not attach.