| Separated DNA strands can reassociate to form duplex DNA
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| Establishing the paternity of children can often be a divisive issue for families. In the past, this was legally settled by establishing the husband of the mother as the biological father. However, with modern DNA technology, paternity can be firmly established. |
| DNA samples should ideally be provided from both parents and offspring. If the DNA of the parent and offspring match, then paternity can be established with greater than 99.999% assurance. If there is no match, then the probability of paternity is 0. |
| The DNA collection method is straightforward. A cotton swab is rubbed on the inside of the cheek to collect epithelial cells, providing a source of the DNA. The DNA is then evaluated for the presence or absence of marker alleles using PCR methods (see Figure 34.11). The FBI uses 13 probes for routine identification of individuals. Even in the case where the father is unavailable or deceased, paternity testing can often be successfully performed. If the father is deceased, a medical examiner's office or hospital may have retained a blood sample that can be used for testing. A personal sample such as a cigarette butt, chewed gum, hair clippings, or electric razor shavings, a licked envelope, or other source of DNA may also be used. The absent parent's DNA type can also be partially reconstructed using that parent's biological parents. |
| Prenatal parentage can also be established after amniocentesis (>12 weeks gestation) or by chorionic villi sampling (CVS) between 9 and 12 weeks gestation. DNA paternity tests have also been legally utilized by persons seeking entry into the US on the grounds that s/he is a biological relative of a citizen, by persons seeking to establish Native American tribal rights, by persons seeking to establish decendency from famous individuals, or by persons seeking to establish biological relationships with long-lost siblings. |
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| Figure 30.3 The structures of different forms of DNA include the B-, A-, and Z-forms. The sugar phosphate backbone of the DNA strands is colored blue. The nucleotide bases forming the internal base pairs are yellow for pyrimidines (thymine and cytosine) and red for purines (adenine and guanine). |
| Knowledge of a person's genetic background provides valuable information for predicting risk for, and susceptibility to, disease for both the individual and his/her descendants. Like a family medical history, this information may be transmitted verbally from previous generations; however this history is often woefully incomplete or even absent in the case of adoptees or children of sperm donors. The process of DNA banking allows an individual to store a sample of DNA for future access. Possible uses include paternity testing, identification of abducted children, and validation of estate claims. |
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| Because the DNA strands are complementary and are held together only by noncovalent forces, they can be separated into individual strands. This strand separation or denaturation of DNA is commonly induced by heating the solution. The dissociation is reversible, and on cooling, the interactions between the complementary nucleotide sequences reassociate or reanneal to reform their original base pairs. This is the basis for one of the primary methods for DNA analysis, Southern hybridization (Chapter 34). Because adenine and thymine interact through two hydrogen bonds and guanine and cytosine through three (Fig. 30.2), AT-rich regions melt at lower temperatures than G:C-rich regions in DNA. The
denaturation of DNA can also be induced locally by enzymes or DNA binding proteins. The promoter region of DNA contains a TATA sequence (the TATA box - see Fig. 33.1), an easily melted region of DNA that facilitates the unwinding of DNA during the early stages of gene expression (Chapter 33).
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| The human genome contains 35 000-40 000 different protein coding genes scattered over 23 chromosome pairs. These different genes represent unique DNA sequences that are present in single copies or at most only a few copies per genome. There are also several types of repeated DNA sequences within the genome. These are divided into two major classes: middle repetitive (<10 copies per genome) and highly repetitive (>10 copies per genome) sequences.
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| Some middle repetitive DNA consists of genes that specify transfer RNAs, ribosomal RNAs, or histone proteins that are required in large amounts in the cell. Other middle repetitive DNA sequences have no known useful function, but may participate in DNA strand association and chromosomal rearrangements during meiosis. The best-characterized repetitive sequence in humans is known as the Alu sequence. Between 300 000 and 500 000 Alu I repeats of about 300 base pairs are scattered throughout the human genome, comprising 3-6% of total DNA. Individual repeats of the Alu sequence may vary by 10-20% in identity. Alu sequences also occur in monkeys and rodents, and similar sequences occur in slime molds and other animal phyla. In addition to the Alu family, there are several other families of middle repetitive DNA, mostly of unknown function, in the human genome.
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| Satellite DNA is highly repetitive DNA that was originally identified because it has a slightly different buoyant density from the main band of DNA when centrifuged through a CsCl gradient. Satellite DNA consists of clusters of short, species-specific, nearly identical sequences that are tandemly repeated hundreds of thousands of times. These clusters are deficient in protein-coding genes and are found principally near the centromeres of chromosomes, suggesting that they may function to align the chromosomes during cell division to facilitate recombination. Because these repetitive sequences cover long stretches of chromosomes (100s to 1000s of kilobase pairs; kbp), determining the sequence of satellite DNA and sequencing the centromere region of DNA are major challenges to completing the sequence of eukaryotic genomes.
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| The nucleus of eukaryotic cells contains the majority of the DNA in the cell - genomic DNA. However, DNA is also found
in mitochondria and in plant chloroplasts, which is consistent with current 'endosymbiont' theories for the origins of these cellular organelles. The mitochondrial genome is small in size, circular, and encodes relatively few proteins. In human beings, the mitochondrial genome encodes 22 tRNAs, 2 rRNAs, and 13 proteins. All of the mitochondrial encoded proteins are involved in the respiratory apparatus. The mitochondrial-encoded proteins are cytochrome b, three subunits of cytochrome oxidase, one of the subunits of ATPase, and seven subunits of NADH dehydrogenase.
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The remainder of the proteins that are found in mitochondria (about 1000) are produced from nuclear genes, synthesized in the cytoplasm on 'free' ribosomes, then imported into the mitochondrion. This import process requires a special N-terminal 'mitochondrial-import' sequence of about 25 amino acids in length that forms an amphipathic helix capable of interacting with receptors on the mitochondrial surface.
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| Those few proteins that are encoded by the mitochondrial genome are also translated on mitochondrial ribosomes that are assembled on the two mitochondrial rRNAs after import of the nuclear-encoded ribosomal proteins into the mitochondria. Because there are only 22 tRNA genes encoded by the mitochondrial genome, yet 61 potential amino acid codons, there has been a dramatic simplification of the genetic code in the mitochondrion. Thus the genetic code of mitochondria has a restricted set of codons, compared to the universal (nuclear) genetic code (Chapter 32).
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