Medical Biology - Genetics 2023-2024 - Chapter VII PDF

Exercise 7. Topic: Introduction to genetics. Parasitological and mycological diagnostics – serological and molecular methods. Glossary: Agglutination – the clumping of bacteria, red blood cells, or other cells, due to the introduction of an antibody. Allosome = heterochromosome - a sex chromosome. A...

Exercise 7. Topic: Introduction to genetics. Parasitological and mycological diagnostics – serological and molecular methods. Glossary: Agglutination – the clumping of bacteria, red blood cells, or other cells, due to the introduction of an antibody. Allosome = heterochromosome - a sex chromosome. Antygen - Any substance that induces the immune system to produce antibodies against it (stimulating an immune response). We can distinguish: heteroantigens -a foreign substance ,originate from outside the body or autoantigens; (self-antigens)- substance originate within the body Antybody – also called immunoglobulin (Ig); a protein produced by lymphoid cell (plasma cell) in response to foreign substances (antigens) and capable of binding especially with its homologous antigen or with substances that are chemically very similar to that antigen. Antigen-antibody reaction – the formation of an insoluble complex between an antigen and its specific antibody. In the case of soluble antigens, the complex precipitates, while cells carrying surface antigens are agglutinated. Autosome – any chromosome other than a sex chromosome. Diploid or diploidy – referring to the situation or state in the life cycle where a cell or organism has two sets of chromosomes: one from mother and one from father. ELISA - Enzyme-Linked Immunosorbent Assay – an immunochemical technique for detecting and measuring antigens or antibodies in a solution; the assay uses enzymes as indicators. Epitope – the part of an antigen molecule (the antigenic determinant): a molecular region on the surface of an antigen capable of eliciting an immune response and to which an antibody attaches itself. FISH - Fluorescent in situ hybridization - is a cytogenetic technique which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity. Haploid or haploidy – referring to the situation or state in the life cycle where a cell or organism has a single set of chromosomes. Heterochromosome – a sex chromosome. Molecular probe - a short fragment of a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome The probes can be labelled fluorescently or with a radioisotope for visualization purposes. Nucleic Acid Hybridization – coupling of nucleic acids with a complementary sequence. This connection (annealing) can take place both between single-stranded DNA and DNA, DNA and RNA, RNA and RNA. Hybridization occurs spontaneously. PCR – Polymerase Chain Reaction – it mimics the DNA replication process in vitro. The method is based on the chain reaction of DNA polymerase as a result of repeated heating and cooling of the sample under laboratory conditions. It allows the amplification of a specific short DNA fragment (several dozen - a few thousand bp). Precipitation – in immunology: is a reaction in which a soluble antibody reacts with a soluble antigen to give an insoluble product or the precipitate. qPCR - quantitative Polymerase Chain Reaction – amplification of specific genomic DNA sequences using fluorochrome-labeled primers and measuring the fluorescence intensity of individual alleles (determining the number of copies of a given sequence in a cell). Serological methods - diagnostic methods based on the examination of the presence of antigens and antibodies in the blood serum. I. Introduction to genetics. 1. Molecular structure of DNA The deoxyribonucleic acid (DNA) acts as a carrier of genetic information of living organisms and some viruses. It consists of 2 polynucleotide chains or strands, wound around each other in such a way that they resemble a twisted ladder. This structure is referred to as the double helix. The backbone of each of these strands is a repeating pattern of a 5-carbon sugar and a phosphate group. Each sugar is attached to one of the four nitrogen-containing bases. The four types of nitrogen bases are adenine (A), thymine (T), guanine (G) and cytosine (C). The sugar present in the nucleotide is a deoxyribose, hence the name deoxyribonucleic acid (DNA). In the double helix DNA structure, all four bases are confined to the inside of the double helix, held in place by hydrogen bonds linking complimentary bases on the two strands. The sugar-phosphate backbones of DNA are on the outside of the double helix. Adenine and thymine are paired by two hydrogen bonds, whereas cytosine and guanine are paired by three hydrogen bonds (complementary base pairing). The bases are stacked up the ladder and the hydrophobic bonding between the bases gives the DNA molecule stability. The two DNA strands in the double helix run in opposite directions (their sugar-phosphate backbones run in opposite directions, one - in the 5' to 3' direction, the other - in the 3' to 5' direction; antiparallel to each other) to help the bases in each base pair fit into the double helix. This means that the nucleotides in each strand of DNA are exactly complementary to those in the other strand. The complementary base-pairing of A with T and G with C enables optimization of energy levels within the double helix. In this double helix arrangement, the width of each base pair remains the same, meaning the same distance is kept between the sugar-phosphate backbones, along the length of the DNA molecule. The twists or turns in the two sugar-phosphate backbones of the double helix occurs every ten base pairs, which maximizes efficiency of the base-pair packing. The two polynucleotide strands of the DNA double helix provide a simple basis for copying the information in the molecule. On separation, each of the two strands serves as a template for creating an exact or identical copy of the DNA molecule. In cell’s nucleus, DNA is packed into tightly coiled structures called chromatin. COMPLEMENTARITY Figure 1.Structure of DNA DNA is present in nuclei and also outside of the nuclei, in the mitochondria of eukaryotic cells as a mitochondrial DNA (mtDNA). This type of DNA is a double-stranded circular form of genetic material found in the mitochondrial matrix. The human mtDNA contains about 16.569 bp and covers 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mtDNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system (NDs, ATP-ases, COXs, Cytb) Figure 2.The human mtDNA structure Molecular structure of RNA The ribonucleic acid (RNA) has all the components similar to those in DNA with only two main differences within it. RNA has ribose sugars connected with phosphate groups and nitrogenous bases: the adenine, guanine, cytosine and uracil (instead of thymine in DNA). Adenine and uracil are considered as the major building blocks of RNA and both of them form base-pair with the help of two hydrogen bonds. RNA carries genetic information from DNA, that is translated by ribosomes into various proteins necessary for cellular processes. There are the three main types of RNA involved in protein synthesis: - mRNA - messenger RNA, that serve as temporary copies of the information found in DNA; - rRNA, - ribosomal RNA, that serve as structural components of protein-making structures known as ribosomes; - tRNA - transfer RNA, that transfer the appropriate amino acid to the ribosome to the end of the growing amino acid chain. Currently, other RNAs that play significant roles (mainly help control gene expression in cells) are also known, e.g.: - microRNA (miRNA); - short interfering RNA (siRNA); - long non-coding RNAs (lncRNA); - small nuclear RNA (snRNA). RNA also occurs as the genetic material of some viruses, single-stranded or double-stranded RNA, analogous to double-stranded DNA. 3. Replication DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules in terms of nucleotide sequence. The precise process of DNA replication (also DNA duplication) is the basis for the transmission of identical genetic information to new cells and generations of individuals (heredity). It occurs during the S phase of the interphase, i.e. the interdivision phase, and its aim is to provide daughter cells with complete genetic information. 4. Chromosome The eukaryote chromosome is a thread-like structure made of an organism's genetic material (a single DNA molecule) and packaging proteins called histones, which, with the help of other proteins, bind to condense the DNA molecule and maintain its integrity. Chromosomes are visible in the cell’s nucleus when the cell is dividing. During the cell cycle, condensation and decondensation of chromosomes occur - morphological changes in the chromatin structure because of the reversible modification of chromosomal proteins. The basic unit of chromatin are nucleosomes - segments of DNA wound (approximately 1.7 turns of DNA) around histone octamers (eight histone proteins) forming core particles, which are connected by stretches of linker DNA. The addition of one H1 histon protein wraps another 20 base pairs, resulting in two full turns around the octamer. Chromatin looks like the beads-on-a-string. The nucleosomes fold up to produce a 30-nm fiber that forms loops, the fiber is compressed and after tight coiling became chromosomal chromatid. The chromosome of eukaryotes consists of two chromatids (sister chromatids) linked together with a centromere - a constriction point, which divides the chromosome into two sections, or “arms.” The short arm of the chromosome is labelled the “p arm” and the long arm – labelled the “q arm.” The location of the centromere on each chromosome gives the chromosome its characteristic shape and can be used to help describe the location of specific genes. Figure 3. Chromosome structure https://ghr.nlm.nih.gov/primer/basics/chromosome Types of chromosomes  Metacentric chromosomes - the centromere in the centre, such that both sections are of equal length. Human chromosome 1, 3, 16, 19, 20 are metacentric.  Submetacentric chromosomes – the centromere situated so that one chromosome arm is shorter than the other. Human chromosomes 2, 4 through 12, 17, 18 and X chromosome are submetacentric.  Acrocentric chromosomes – the centromere which is severely offset from the centre leading to one long and one very short arm. Human chromosomes 13, 14, 15, 21, 22 and Y chromosome are acrocentric.  Telocentric chromosomes - the centromere at the very end of the chromosome. Humans do not possess telocentric chromosomes (only formed through cellular chromosomal errors); found in other species such as mice. II. Serological and molecular methods in diagnostic 1. Serological methods Introduction Serological diagnosis is usually based on either the demonstration of the presence or concentration of specific antibodies (Ab) or specific antigens (Ag) in the evaluated organism (e.g. In blood serum, in feces). Such biochemical test are named an immunoassay (IA) method. Antibodies are powerful research tools because they bind specifically to a unique epitope on the antigen, thereby allowing the detection of a specific protein in an assay while avoiding detection of unrelated proteins. Each antibody consists of four polypeptides – two heavy chains (H) and two light chains joined to form a "Y" shaped molecule. The antibody contains a constant region common to all antibodies produced by a particular species and a variable region that is unique and specific to a particular epitope (Figure 4). Human antibodies are classified into five isotypes (classes) according to their H chains, which provide each isotype with distinct characteristics and roles. (Table1). Figure 4. Structure of antibody. A variety of methods have been developed for visualizing the primary Ag-Ab reaction. With naked eye precipitation of Ag-Ab complexes can be visible (e.g., immunodiffusion and immunoelectrophoresis). But now the most of immunoassays use a variety of labels, typically chemical-linked or conjugated to the desired antibody or antigen, e.g.: enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA). The immunohistochemistry allows for the localization of a specific protein within a cell or tissue and immunoprecipitation allows for the isolation of a specific protein from within a mixture of proteins. Table 1. Types of antibodies. Structure Properties IgG Secreted in the plasma cell in the blood. Able to cross placenta into the fetus. IgM May be attached to the surface of a B cell or secreted into the blood. Responsible for early stages of immunity IgA Found in saliva, tears and breast milk. Protect against pathogens. IgD Part of B cell receptor. Activates basophiles and mast cells. IgE Protect against parasitic worms. Responsible for allergic reactions. Serological methods in parasitological and mycological diagnostics. In recent years, the use of serological methods has broadly entered the routine diagnosis, also mycological/parasitological diagnosis. These methods are relatively easy to make and very fast compared to culture methods. Serological methods should be used:  in cases of suspected chronic and systemic fungal infection, especially in patients with hematologic malignancies.  in each case of suspected parasitosis, especially parenteral, as additional diagnostics when the test result of the coproscopic study is negative  when microscopic examination is impossible.  to confirm parasitosis imported from tropical or subtropical countries The most frequently used serological markers of fungal infections and parasitic infections presents Table 2. Table 2. Serological markers of fungal infections and parasitic invasions Type of Detected element Species /Genera infection Fungal components of the fungal cell wall, released during growth, detected in body fluids Parasitic specific antibodies detected in body fluids galactomannan Aspergillus mannan; Candida glucuronoxylomannan Cryptococcus class IgM – early tick-borne infection invasion class IgG – elder Toxoplasma; Giardia; Entamoeba; invasion Cryptosporidium; Toxocara; Ascaris, Echinococcus; Taenia solium; Trichinella class IgA Toxoplasma; Toxocara antigen Em 1 Echinococcus granulosus i E. multilocularis antigen Em 2 Coproantigen – detected in parasitic diseases of the gastrointestinal tract Echinococcus multilocularis Giardia intestinalis, Cryptosporidum parvum, Entamoeba histolytica Serological methods, which have been applied in mycological and parasitological diagnosis can include: - latex agglutination test   reaction of the test component (antibody or antigen) with the detection component  direct agglutination – antigen detection,  indirect agglutination – antibody detection, diagnostic applications:  detection of Candida, Aspergillus and Cryptococcus:  detection of Leishmania in the urine; - immunofluorescence tests  use of antibodies labelled with fluorescent dyes: fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TRITC):  diagnostic applications:  detection of Candida, Aspergillus, Alternaria, Pneumocystis,  detection of Leishmania, Giardia, Cryptosporidium, Entamoeba, Plasmodium, Trypanosoma, Echinococcus, Toxocara, Ascaris, Schistosoma;  - ELISA immunoenzymatic assay – the use of antibodies labelled with an enzyme (e.g. horseradish peroxidise) detection of colour change as a result of the reaction of the enzyme with the substrate,  diagnostic applications:  detection of Candida, Aspergillus: Platelia Candida ELISA Ag Plus - is a one-step enzyme immunoassay using a sandwich microplate for detecting the circulating mannan Candida antigen in human serum or plasma. Platelia™ Aspergillus Ag – gallactomann - microplate sandwich test for detection galactomannan Aspergillus antigen in serum samples from adult and pediatric patients; and samples of bronchoalveolar lavage (BAL).  detection of protozoa: Toxoplasma (IgA, IgG), Giardia (IgG), Entamoeba (IgG), Cryptosporidium (IgG),  detection of helminth: Echinococcus (Em2+, Em1,IgG), Taenia solium (Trichuris, IgG), Trichinella (IgG), Dirofilaria, Toxocara (IgA, IgG); - WESTERN BLOT  separation of proteins in the polyacrylamide gel (SDS-PAGE), then transferring them to a nitrocellulose membrane and binding to antibodies from the test serum,  diagnostic applications:  detection of Candida, Aspergillus, Cryptococcus, Histoplasma,  detection of Toxoplasma (mother and newborn serum, IgG), Giardia, Entamoeba, Cryptosporidium, Echinococcus, Taenia solium (cystiscercosis),  detection of tick-borne diseases (Lyme boreliosis) – the time of infection can be estimated based on the antibody concentration and the IgM to IgG ratio (Figure 3). Level of produced antibodies Early phase( IgM) Late phase(IgG) 4-6 weeks 6-9 weeks months Figure 3. Production of IgM and IgG antibodies in the course of Lyme boreliosis. years Serological tests allow for quick diagnosis of many infectious and invasive diseases of man, however in some cases false-positive or negative results may be obtained (Table 3). Table 3. Advantages and disadvantages of serological methods. Advantages of serological methods Disadvantages of serological methods antigen detection at an early stage of  false positive or false negative results infection/infection  elimination of antibodies from the bloodstream  fast detection  cross reactions  high sensitivity  antigenic similarity between microorganisms  high specificity  lack of antibodies in immunocompromised people  2. Molecular methods. Molecular methods described below are used in microbiological diagnostics (virology, bacteriology, mycology) and parasitological diagnostics of humans and animals. They are also used in the genetic diagnosis of hereditary diseases and human cancers. The element analysed in the molecular methods is the evaluation of the presence of DNA sequences (or RNA in some methods) in the tested material characteristic of the sought species of the microorganism suspected of causing infection in the tested material. Sequences characteristic of the species sought are referred to as molecular probes. Molecular diagnostics is based on the search for specific sequences in the genetic material which can be:  highly variable areas – minisatellite and microsatellite sequences:   differentiation of strains or individuals; moderately variable sequences – internal transcribed spacer (ITS), mitochondrial genes (cytochrome oxidase subunit):   species differentiation; conservative sequences – DNA of a small subunit of ribosomal RNA, genes encoding proteins:  higher taxonomic levels. Indications for the use of molecular methods:  cases of severe systemic fungal infections;  when results of cultures and microscopic examination are negative;  investigation epidemiological outbreak of nosocomial infection;  low intensity of infection, below the threshold detected microscopically;  differentiation of similar species;  confirmatory tests;  determination of drug resistance in the parasite. Molecular methods can be initially divided into: 1. Methods that do not require DNA multiplication – based on chromosome sequence hybridization (specific binding) of a probe containing a fluorescent label – fluorescent in situ hybridization (FISH) (Table 4). Micro-chip methods using specific gene probes, usually fluorescent. 2. Methods based on initial multiplication/amplification of DNA in the PCR reaction or its variations used to multiply the analysed sequence. This amplified sequence can then be analysed using restriction enzymes (Table 5). Table 4. Methods based on nucleic acid hybridization without DNA/RNA amplification. Methods without preliminary DNA multiplication Application in diagnostics nucleic acid hybridization Dot-blot the tested samples are dotted on the membrane, the  result in the form of coloured dots yeast from genera: Saccharomyces, Debaryomyces, Kluyveromyces  identification of phytopathogens  detection of Plasmodium, Leishmania  detection of Echinococcus (tapeworm) copraantigens in dogs Southern-blot It is based on detecting DNA fragments using DNA  diagnosis of invasive candidiosis and aspergillosis probes. Stages:  detection of Plasmodium, Leishmania It is based on the detection of RNA fragments (most  detection of fungal allergens (e.g. Alternaria) commonly mRNA) using DNA or RNA probes. Steps:  detection of Aspergillus sp.  DNA isolation,  gel DNA separation,  then membrane transfer and hybridization with the probe. Northern-blot  RNA isolation,  detection of Plasmodium, Leishmania, Trypanosoma  RNA separation on the gel under  determining drug susceptibility of parasites denaturing conditions,  then membrane transfer and hybridization with the probe. hybridization of nucleic acids to probes FISH (in-situ The fluorochrome labelled probe is added directly to  detection of Candida, Cryptococcus, Histoplasma hybridization) the sample without first isolating DNA or RNA  detection of invasive infections induced by Plasmodium, Leishmania, Trypanosoma in the blood and cerebrospinal fluid  detection of Cryptosporidium, Giardia, Microsporidium Table 5. Methods based on initial multiplication / amplification of DNA by PCR or its variants. Methods based on initial multiplication of DNA Stages of the analysis PCR - polymerase chain Mimics the process of DNA replication in vitro. 1. DNA isolation reaction It allows you to amplify a specific fragment of DNA (several tens - 2. PCR - amplification several thousands bp). The method is based on the chain reaction 3. Product distribution and of DNA polymerase as a result of repeated heating and cooling of visualization on agarose gel the sample under laboratory conditions. PCR in diagnostics  diagnostics of Candida, Aspergillus, Pneumocystis, Fusarium, Cryptococcus  phylogenetic studies in mycology  differentiation of Echinococcus, Schistosoma, Opistorchis, Ancylostoma, Necator  detection of Echinococcus, Schistosoma, Trichobilharzia, Toxocara in environmental studies RT-PCR - reverse transcription The template is RNA, which in the process of reverse transcription 1. RNA isolation polymerase chain reaction is transcribed into DNA and then the DNA is amplified by PCR 2. Reverse transcription 3. PCR - amplification 4. Product distribution and visualization on agarose gel RT-PCR in diagnostics  diagnostics of Candida albicans  detection of Fasciola hepatica from the environment  detection of Babesia nested PCR two different starter pairs - "external" and "internal" 1. DNA isolation two PCR reactions: 1. with "external" primers, 2. with 2. I PCR - amplification "internal" primers; a first reaction product forms a matrix with external primers in the second reaction 3. II PCR - amplification the sequence reproduced using the "internal" primers with internal primers lies within the sequence reproduced with the use of 4. Distribution of II PCR "external" primers products and visualization increased sensitivity and specificity of the reaction in a polyacrylamide gel nested PCR in diagnostics  diagnostics of Pneumocystis  detection of Candida, Cryptococcus, Aspergillus, Penicillium  detection of Giardia Multiplex PCR More than one pair of primers are used in one PCR reaction 1. DNA isolation simultaneously -> it allows for the multiplication of more than 2. PCR - amplification with one sequence, e.g. amplification of three different genes multiple primer pairs quick analysis of large areas of DNA - saving time and work simultaneously reaction products must vary in length 3. Product distribution and used in affinity and forensic analysis visualization on agarose gel Multiplex PCR in diagnostics  detection of Candida, Aspergillus, Cryptococcus  differentiation of Taenia solium, T. saginata, Trichinella sp.  enables the diagnosis of several pathogens at the same time Methods for detection of mutations defined Real-time PCR It is a method of observing the amplification in real time using 1. RNA isolation fluorescent probes or markers staining nucleic acids (e.g. SYBR 2. amplification with fluorescent Green) probes - observation of the With the arrival of PCR products, the intensity of fluorescence product's growth in real time - recorded by the detector increases after each reaction of the cycle Real-time PCR in diagnostics  detection of Candida (LightCycler), Aspergillus fumigatus  diagnostics of Toxoplasma gondii, Giardia (from the environment), Plasmodium, Cryptosporidium, Trypanosoma  differentiation of Neospora, Trichinella, Dirofilaria Table 6. Advantages and disadvantages of molecular methods. Advantages Disadvantages  the possibility of using different materials  frequent false negative results  small amount of material needed for  too high sensitivity testing  lack of standardization (lack of standardization of DNA  speed of obtaining the result  high sensitivity isolation methods)  easy contamination of samples  high cost Components of the PCR reaction 1. 1. DNA polymerase - an enzyme that has the ability to synthesize a complementary strand based on a single-stranded DNA chain - catalyzes the reaction, 2. buffer - provides the appropriate reaction environment for the enzyme (pH, ionic strength), 3. Mg2+ – cofactor of DNA polymerase, 4. dNTP – triphosphates of all four deoxyribonucleosides: dATP, dCTP, dGTP, dTTP - substrates for the construction of new DNA strands, 5. DNA – a template for the construction of new strand (template DNA can be isolated from any kind of organism, whether genomic DNA, mitochondrial plasmid and cDNA transcribed from mRNA). 6. primers - chemically synthesized (on request) DNA sequences 15-30 bases long that surround the amplification site, complementary to the DNA strand. Thanks to the starters, the DNA polymerase can start synthesizing the complementary strand. An important feature is having a free 3' end for the synthesis of new strands by DNA polymerase, 7. Deionized water - it must be free of enzymes that digest DNA (DNAz) or biological impurities. The PCR reaction due to its features is one of the basic tools for work in a molecular laboratory. Visualization of the PCR product: the simplest way is to separate the products in an agarose or polyacrylamide gel using dyes: ethidium bromide, Midoria Green, silver salts that attach to the DNA sequence. After gel separation, the gel is placed e.g. on a transilluminator (emitting light in the ultraviolet spectrum) and the bands with the PCR product are visualized. Table 7. The course of the PCR reaction Each polymerase chain reaction consists of several cycles of successive steps: 1. Denaturation -heating the sample to 95° C in order to melt the double helix (denaturation), after which each of the strands acts as a template. Breaking hydrogen bonds 2. Annealing - lowering the temperature to attach the primer sequences to the DNA strand (annealing = hybridization of the primer sections). The temperature that is used in the annealing step depends on the length of the primers and the content of GC pairs To polymerase could synthesize a complementary strand of double-stranded section is required primer. 3. Extension/elongation – synthesis of a complementary strand by a polymerase. The right sequence amplification process. https://www.quimigen.com/comprar/cat-conventional-pcr3473.html The control of the amplification process consists in the proper selection of temperatures and number of repetition cycles. During the reaction, no components are added to the reaction. The product of one reaction cycle is used as a template in subsequent cycles - thus determines the chain reaction. We observe an exponential amplification of exponential products. https://commons.wikimedia.org/wiki/File:PCR_basic_principle1.jpg licencja: GFDL + CC-by-sa Table 8 Advantages and disadvantages of PCR. Advantages  high sensitivity - possible duplication of a single Disadvantages  the risk of contamination – with e.g. the genetic material of the person performing the DNA molecule, analysis, or in the case of microbiological analyses, the possibility of bacterial contamination from the immediate vicinity of the laboratory  selectivity - only the selected sequence is  the need to work in sterile conditions  the need to select the appropriate primers for the reproduced    rate - the test gene amplification time of 2-3 hours high yield - 106 - 109 copies of the tested gene as a result of one reaction simplicity of implementation reaction - knowledge of the gene sequence  the need to perform additional analyses to visualize the obtained effects (gel separation, restriction enzyme digestion, staining) References: 1.Drewa G., Ferenc T. Genetyka medyczna, Elsevier Urban&Partner, Wrocław, 2011; Rozdział 3 – Kwasy nukleinowe; Rozdział 27 – Metody molekularne badania genomu 2. Jorde L.B.; Carey J.C.; Bamshad M.J.; White R.L.: Medical Genetics, Third Edition 2006 3. Campbell, J.B. Reece: Biology. Pearson, Benjamin Cummings, Seventh Edition 2005