Molecular Techniques

Basic Terms and Principles

The human genome is distributed among 46 chromosomes , 22 pairs of autosomal chromosomes, and 2 sex chromosomes. Biochemically, the genetic information is stored in the DNA , which is a sequence of 3 billion nucleotides . Nucleotides consist of one of four bases (A, adenine ; T, thymine ; C, cytosine ; G, guanine ) and a sugar-phosphate backbone . Two complementary, anti-parallel DNA strands are bound together by hydrogen bonds to form a double helix structure ( Fig. 30.1A ). The hydrogen bonding is responsible for the pairing of the complementary bases in the DNA: A binds T, and C binds G. As a result of this complementarity, all the information in the DNA helix is duplicated on each strand, which is vital for DNA transcription, DNA replication, and DNA repair. The complementary nature of base pairing is also the underlying principle for the majority of molecular methods such as the binding of complementary primers in PCR assays, the hybridization of complementary fluorescence-labeled probes in FISH assays, and the binding of complementary nucleotides in sequencing assays.

The DNA is organized into linear domains of genes, functional RNAs, regulatory elements, and repetitive sequences. Genes are the molecular units of heredity, encode proteins, and consist of many structural elements of which the protein coding sequence is often only a small part ( Fig. 30.1B ). The transcription of genes is performed by an enzyme termed RNA polymerase , which binds to the promoter region and produces a pre-messenger RNA ( mRNA ), whose nucleotide sequence is complementary to the DNA, except that the base uracil (U) replaces thymine (T). To control the amount of mRNA, the transcription is tightly regulated by promoter , enhancer , and repressor elements that bind numerous regulatory proteins, such as transcription factors . The introns are then removed from the pre-mRNA and the exons are spliced together to the mature mRNA. The translation into a protein begins at the mRNA's start codon , ends at the stop codon , and is carried out by ribosomes that add new amino acids to the growing polypeptide chain . The genetic code is read in three nucleotides ( codons ) at a time; each codon on the mRNA interacts specifically with three complementary bases ( anti-codon ) on specialized RNA molecules, termed transfer RNA (tRNA), which are covalently attached to the amino acid specified by the anti-codon. When the tRNA's anti-codon binds to the complementary mRNA's codon, the ribosome attaches its amino acid cargo to the new polypeptide chain. The translation, stability of the mRNA, and consequently the number of proteins are regulated by the 5′ and 3′ untranslated regions .

Mutations are permanent changes in the nucleotide sequence of the DNA and usually result from DNA damages that are not repaired. Mutations can have either no effect (silent) or can change the function of a protein ( loss-of-function mutation , gain-of-function mutation ). Mutations within genes that affect one or few nucleotides are usually classified as synonymous mutations , which code for the same amino acid and do not change the protein function; or as non-synonymous mutations, which change the amino acid sequence and regularly alter the protein function. Non-synonymous mutations can be further divided into missense mutations , resulting in the incorporation of a different amino acid; nonsense mutations , resulting in a premature stop codon and leading to a truncated protein; and insertions and deletions that add or remove one or more nucleotides, respectively, and may cause a shift in the reading frame (therefore also termed frameshift mutations ). Large genomic aberrations that affect chromosomal structures are classified as copy number gains (amplification), which increase the dosage of the genes located within the alteration; copy number loss (deletion), which lead to a loss of genes in the affected regions; and fusion genes , which result from translocations , inversions , or interstitial deletions and juxtapose previously separated DNA regions next to each other.

Polymerase Chain Reaction

Endpoint Polymerase Chain Reaction

The polymerase chain reaction (PCR) has revolutionized the entire field of molecular biology by allowing a fast, cheap, specific, and sensitive amplification of individual DNA fragments from a complex pool of DNA. PCR-based methods are an indispensable component for a plethora of molecular biologic techniques, and are widely used to detect pathogens, to sequence genes, and to quantify gene expression.

PCR-based assays require four basic components: (1) template DNA; (2) the four nucleotides found in the DNA; (3) primers, short DNA fragments with a defined sequence complementary to the target DNA to be amplified; and (4) the DNA polymerase , an enzyme that connects the nucleotides to the PCR product, termed amplicon . A mix of these components in an adequate buffer is then placed in a thermal cycler , which is a machine that raises and lowers the temperature in three predefined steps ( Fig. 30.2A ):

• 1.

  Denaturation: The DNA double helix separates to two single strands.

• 2.

  Annealing: The primers specifically bind to the single-stranded DNA fragments that are complementary to their own nucleotide sequence. The DNA polymerase binds to the DNA-primer site.

• 3.

  Extension: The DNA polymerase synthesizes the new DNA strand by adding nucleotides that are complementary to the DNA template strand.

With each cycle of these three steps, the number of copied DNA molecules doubles, so that billions of amplicons are generated after 20 to 40 PCR cycles (see Fig. 30.2B ). The amplicons are then visualized with agarose gel electrophoresis, which separates DNA products on the basis of size, or are further analyzed in various downstream application such as DNA sequencing.

PCR is a very sensitive method, and very prone to contamination. Quality controls, including negative and positive controls, are therefore essential. The most common and serious problem is carryover contamination of amplicons from previous PCR reactions. For this reason, pre- and post-PCR areas should be spatially separated in every lab. Other difficulties in performing PCR result from low-quality DNA (often caused by inappropriate fixation of the specimens, e.g., unbuffered formalin), unspecific PCR products due to nonspecific binding of the primers, or incorporation of incorrect nucleotides by the DNA polymerases.

In addition to the traditional PCR technique described above, various other PCR approaches are based on the same principle with slight modifications. In nested PCR assays, the sensitivity and specificity of PCR is further increased by amplifying the initial amplicon with a new set of primers intrinsic to the first set of primers. Multiplex PCR assays use two or more pairs of primers targeting different DNA regions in the same reaction mix, resulting in multiple amplicons.