2.2 Types of polymorphism

Length polymorphisms (loci) are repetitive tandem sequences and individual alleles (gene forms) that differ in the number of repeats of a sequence motif. These loci are widespread throughout the human genome and show sufficient variability among individuals in a population. They have become important in several fields including genetic mapping; linkage analysis; and human identity testing. These tandemly repeated regions of DNA are typically classified into several groups depending on the size of the repeat region such as variable number of tandem repeats (VNTRs) and short tandem repeats (STRs). The other types of polymorphisms are short insertions and deletions (InDels) or single-nucleotide polymorphisms (SNPs).

#### 2.2.1 VNTR

VNTR polymorphisms are minisatellite DNAs with a total sequence length of up to 1000 base pairs and with a repetitive sequence motif of more than 6 base pairs. The benefit of VNTR analyses by PCR is a high degree of discrimination and unlimited quantitative determination, but, on the other hand, the disadvantage is the low sensitivity of the method (ranges from 1 to 5% depending on the analyzed VNTR locus and the combination of recipient-donor allele pair). The PCR products

(VNTR alleles) are visualized by fragment analysis via agarose gel electrophoresis, and when the donor's and the recipient's genotypes are detected in the sample together, the level of chimerism is determined densitometrically.

#### 2.2.2 STR

STR polymorphisms are the repetitive sequences of microsatellite DNAs composed of up to one to six base pairs [12]. However, the most common STRs can reach, in their final range, from 100 to 500 base pairs [13]. The number of STR repetitive units may vary widely in populations. There are literally hundreds of STR systems which have been mapped throughout the human genome [14]. These STR loci are found on almost every chromosome. Since 1997, the National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database [15] commonly referred to as STRBase. This STRBase includes used resources and the summary of human STR polymorphisms, their basic information, chromosomal location, overview of alleles, population studies, or available commercial kits. Observed alleles and annotated sequences for each STR locus are described along with a review of STR analysis technologies [16]. STR markers show a high level of polymorphism and consequently provide a significant degree of dissimilarity between individuals [17]. At present, they are considered to be the most informative genetic markers in the characterization of biological material. STR analysis is a very robust method with a sensitivity of 0.4– 1% of minor genotype. Compared to VNTR, this method uses smaller quantities of DNA, including degraded DNA. STR polymorphisms may be amplified using a variety of PCR primers. Nowadays, a lot of multiplex kits are available on the market for human genotyping. The method is based on selective PCR of DNA segments with examined polymorphisms. PCR primers are fluorescently labeled. Separation of the fragments takes place subsequently in the genetic analyzer by capillary electrophoresis, as the carrier medium is used a polymer. In the genetic analyzer, fluorophores are excited by a laser ray to label DNA fragments. Fluorophores absorb laser light and emit light at a longer wavelength. Using an optical system, the emitted light is detected and recorded on a charge-coupled device (CCD) chip. STR alleles are visualized by specific software-like peaks. The area or height of peaks is used for quantification.

#### 2.2.3 InDel and SNP

InDels are biallelic polymorphisms classified among small genetic variations, measuring from 1 to 10,000 base pairs in length [18, 19].

SNP is the smallest possible change in DNA sequence in individuals of a given population. SNPs are most often formed by a point mutation mechanism that is substitution-like, less often by deleting or inserting at a particular DNA site. The distribution of SNPs in the genome is not homogeneous. More often, they occur in noncoding regions, on average 1 of 1000 bp in any selected region of the genome.

SNP and InDel analysis are performed by real-time quantitative polymerase chain reaction (RQ-PCR) with specific primers designed to contain appropriate insertions or deletions or point-to-point substitutions. Real-time PCR allows accurate quantification of amplified products by detection of fluorescence released during the exponential phase of the reaction. This method can use the non-specific intercalating fluorescent DNA-binding dye SYBR Green that binds all doublestranded DNA [20]. More often TaqMan technology is used [21], which utilizes a fluorescent-labeled target-specific probe resulting in an increased specificity and

Suitable Molecular Genetic Methods for the Monitoring of Cell Chimerism DOI: http://dx.doi.org/10.5772/intechopen.88436

sensitivity compared to the SYBR Green method. Additionally, a variety of fluorescent dyes are available so that multiplex PCR can be used to simultaneously amplify and detect many sequences. The TaqMan probe consists of a fluorophore covalently attached to the 5<sup>0</sup> end of the oligonucleotide and the quencher at the 3<sup>0</sup> end. If the probe is in an intact state, even when the probe is hybridized to the target sequence, the emitted fluorescence is suppressed by the quencher. Only during the elongation phase, when the Taq polymerase with its 5<sup>0</sup> exonuclease activity degrades the specifically bound TaqMan probe complex and releases the fluorophore from the quencher, can fluorescence occur and then be measured. The fluorescence intensity is directly proportional to the amount of PCR product. The calculation of the relative amount of target DNA in a sample is done by comparing the Ct values (the threshold cycle, at which the emitted fluorescent signal exceeds the statistical significance level). The resulting portion of the gene of interest (GOI) is calculated by the ΔΔCT method [22]. Data are normalized with the reference (housekeeping) gene, and the amplification and detection of the GOI and the reference gene can be analyzed as a singleplex or multiplex reaction. The multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for an accurate assessment [23]. Multiplex RQ-PCR in a routine practice enables an increase in throughput and reliability, with a reduction in pipetting errors. The sensitivity of this method is about 0.01% of the minor genotype. Due to the influence of the so-called Monte Carlo effect (a greater degree of random effect on very low percentages), it is appropriate to define the lowest significant detection limit of the method. On the other hand, the RQ-PCR is suitable to use for the quantification of up to about 10% of the minor genotype, since it has a lower accuracy at higher percentages.
