Molecular Biology and Genetics

Molecular Diagnostics

• Molecular tests diagnose infectious diseases, ensure accurate and timely diagnoses, initiate proper treatments, and prevent contagious disease spread.

Leading Uses for Nucleic Acid-Based Tests

• Nucleic acid-based tests are used in antimicrobial resistance detection.
• They are used for nonculturable agents such as HPV and HBV.
• They are used for fastidious, slow-growing agents like Mycobacterium tuberculosis and Legionella pneumophila.
• Tests may detect highly infectious agents dangerous to culture, e.g. Francisella tularensis and Brucella species.
• They are used for in situ detection of infectious agents such as Helicobacter pylori and Toxoplasma gondii.
• Tests can detect agents present in low numbers such as HIV in antibody-negative patients and CMV in transplanted organs.
• They identify organisms present in small volume specimens like intra-ocular fluid and forensic samples.
• Tests differentiate antigenically similar agents and are important for detecting specific virus genotypes associated with human cancers like Papilloma viruses.
• They are applied to antiviral drug susceptibility testing to help decide anti-viral therapy for HIV infections.
• They are used for non-viable organisms, which are organisms tied up in immune complexes.
• Can identify point sources for hospital and community-based outbreaks in molecular epidemiology for virulence prediction.

Nucleic Acid Molecular Techniques

• Direct probe testing is better for identification than detection because it is not as sensitive as amplification methods.
• Amplification methods improve the sensitivity of the nucleic acid testing technique.
• Types of amplification include target, probe, and signal amplification, and combinations thereof.

PCR Components

• DNA Sample
• Primers
• Nucleotides (C, G, A, T)
• Taq Polymerase
• Mix Buffer
• PCR Tube

PCR Process (One Cycle)

• Denaturing at 95°C separates the strands.
• Annealing at 55°C allows primers to bind to the template.
• Extension at 72°C synthesizes the new strand.

Human Genetics

• Phenotype refers to observed physical and functional traits.
• Genotype refers to a complete set of genes and alleles.
• Alleles are different versions of homologous genes, like B and b.
• Somatic cells are diploid.
• Somatic cells form the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell.
• Gametes are haploid.
• Gametes only have one set of chromosomes.
• Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the seminiferous tubules of the testis.
• Oogenesis, or oögenesis, is the differentiation of the ovum into a cell competent to further develop when fertilized.
• Meiosis is a special type of cell division.
• Meiosis reduces the chromosome number by half, creating four haploid cells, each genetically distinct from the parent cell that gave rise to them.
• Cells contain 2 copies (alleles) of each gene
• Alleles separate during gamete formation (meiosis)
• Gametes carry only one copy of each gene
• Mendel's first law is also known as the law of segregation.
• The law of segregation states that, 'the alleles of a given locus segregate into separate gametes.'
• Punnett squares show parental gametes and the genotypes of the next generation.
• Homozygous: BB and bb
• Heterozygous: Bb
• Where genes for different traits separate independently into gametes
• Mendel's principles account for chromosome behavior.
• Genes on the same chromosome tend to be inherited together and are called linked genes.
• Crossing over produces gametes with recombinant chromosomes.

Variations On Mendel's Principles

• Incomplete dominance is when an offspring has an intermediate trait of its parents.
• A recessive allele will not create a phenotype if a dominant allele is present.
• An individual must have two copies of a recessive allele to express the phenotype.
• In a dominant/recessive relationship between two alleles, the recessive allele's effects are masked by the dramatic effects of the dominant allele.
• Some genes have more than two alleles in a population.
• Family pedigrees track of genetic traits in humans.
• Pedigrees can determine patterns of inheritance and individual genotypes.
• Most mutations usually involve recessive alleles.
• Examples of recessive disorders: Phenylketonuria, PKU; Tay-Sachs disease; Cystic fibrosis.
• A few disorders caused by dominant alleles include achondroplasia and Huntington's disease.

Sex-Linked Disorders

• Affect mostly males
• Most sex-linked human disorders are due to recessive alleles.
• An example being hemophilia in blood.
• These traits mostly appear in males.
• Tetrads may have sex-linked disorders due to most of the recessive alleles being passed down.
• If a male receives a single X-linked recessive allele from his mother, he will have the disorder.
• Females must receive the allele from both parents to be equally affected.
• A high incidence of hemophilia has plagued the royal famiiles of Europe.
• Codominance is a relation between versions of a gene with multiple alleles.
• Pleiotropy is when 1 gene interferes with 2 other unrelated phenotypes.
• Polygenic traits
• Sex-linked genes
• Environmental effects
• Accidents during meiosis can alter chromosome number.
• Abnormal chromosome count is a result of nondisjunction.
• homologous pairs fail to separate during meiosis I

The Impact of Meiosis

• Sister chromatids can fail to separate during meiosis II.
• An extra chromosome 21 causes Down syndrome.
• The chance of having a Down syndrome child goes up with maternal age.
• Fetal testing identifies inherited disorders early in pregnancy.
• Karyotyping and biochemical tests of fetal cells help people make informed reproductive decisions.
• Fetal cells can be obtained through amniocentesis.
• Examination of the fetus with ultrasound is helpful.
• PGD - Analysis of embryos from in vitro fertilization (IVF) before inserting into womb

DNA Review

• In double stranded linear DNA, 1 end of each strand has a free 5' carbon and phosphate and 1 end has a free 3' OH group.
• The two strands are in the opposite orientation to each other (antiparallel).
• Adenines always basepair with thymines (2 hydrogen bonds) and guanines always basepair with cytosines (3 hydrogen bonds)

DNA Details

• Forces stabilizing DNA structure are Watson-Crick-H-bonding and base stacking.
• (planar aromatic bases overlap geometrically and electronically
• increase energy gain)
• Genomic DNAs are large molecules.
• DNA may be studied in Eschericia coli.
• Some DNA molecules (plasmids) are circular and have no free ends, e.g. mtDNA bacterial DNA which is only 1 chromosome.
• Percentage of non-coding DNA varies greatly among organisms.

RNA

• RNA is ribonucleic acid
• Examples include messenger RNA (mRNA) and transfer RNA (tRNA)

Acids and Proteins

• The "central dogma" of modern biology: DNA RNA Protein
• Transcription is using chromosomal DNA to make RNA for molecule mobility.
• Translation is assembling the protein at the ribosome.
• Bases are read in groups of 3 (= a codon)
• the code consists of 64 codons (43 = 64)

Genetic Code Details

• Code features protein synthesis. 20 amino acids 3 stop codons
• AUG (methionine) is the start codon (also used internally)
• The code is non-overlapping and punctuation-free
• The code is degenerate but NOT ambiguous: each amino acid is specified by one codon
• The code is universal

Reading Details

• A reading frame is not always easily recognizable
• Each strand of RNA/DNA has three possible starting points .
• Up to 30% of mutations causing humane disease are due to premature termination of translation (nonsense mutations or frameshift)
• Insertions or deletions induce mutations in RNA sequence.

Mutations Explored

• Mutation: any heritable change in DNA
• Spontaneous mutations: occur for unknown reasons
• Induced mutations: exposure to substance (mutagen) known to cause mutations, e.g. X-rays, UV light, free radicals
• Mutations may influence one or several base pairs
• Nucleotide substitutions are called point mutations.
• One to many bases can be involved in insertion or deletion.
• They are frequently associated with repeated sequences known as hot spots.

Mutations and Phenotype Variation

• Silent mutations do not alter amino acids.
• Mutations can cause a change in amino acid, be neutral, and remain functional, or be missing.
• If they are missing, protein loses some functions as in sickle cell anemia through mutation.
• Sense mutations involve a change from a termination codon to one for amino acid.
• Nonsense mutations create premature termination codon. Many characteristics can change and give rise to new ones, leading to new potential dangers.

Protein Analysis

• Mutation rates are usually given for specific loci, such as in sickle cell anemia. Protein is sequenced using amino acids joined by peptide bonds. Also, side chains play an important role.
• Interactions that occur between the C=O and N-H groups on amino acids
• Much of the protein core comprises a helices and ẞ sheets, folded into a three-dimensional configuration:.

Advantages of Using a Molecular Test

• High sensitivity allows for theoretical detection of a single organism.
• High specificity allows detection of specific genotypes, determination of drug resistance, and prediction of virulence.
• Offers quicker results than traditional culturing for certain organisms.

Disadvantages of Using a Molecular Test

• They are expensive
• So specific that must have could have good clinical data to support infection by that organism before testing is initiated.
• New organisms will be missed
• Testing may be a problem with mixed cultures.
• Assays are needed for all organisms causing the infection.
• Are they too sensitive that they are clinically relevant?

Explore fundamental concepts in genetics, including mutations, DNA structure, and the genetic code. Understand the mechanisms of mutation, DNA base pairing rules, and the components of nucleotides. Learn about the genetic code and its role in specifying amino acids.