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GENETICS: Case 1 and Case 5 Adkison and Brown Molecular Genetic Techniques A physician should be able to interpret fundamental genetic data just as they interpret biochemical data, ECGs, or respirometry results. To do so, an understanding of basic molecular genetic methodologies is necessary. Below, these fundamental approaches and applications to molecular diagnostics are described. Nucleic Acid Visualization Patient cells are necessary to initiate molecular genetic analysis. Typically, patient material is a blood sample, biopsy specimen, and amniotic fluid or chorionic villus sample, though any nucleated cell sample is acceptable. Using blood as an example, total genomic DNA is extracted and the DNA is visualized and/or prepared for subsequent analysis by a number of techniques including restriction fragment length polymorphism (RFLP) and Southern blotting, DNA amplification using the polymerase chain reaction (PCR), or DNA sequence analysis. RFLP and Southern Blot Analysis Southern blotting is a method to visualize DNA of interest. There are three fundamental elements to the Southern blot procedure: (a) fragmentation of the DNA, (b) separation of the DNA on the basis of fragment size, and (c) identification and visualization of informative fragments using a probe. Fragmentation typically occurs through the use of restriction endonucleases, which are also referred to as restriction enzymes. Restriction endonucleases are enzymes of bacterial origin that bind to the DNA at specific sites and cleave both strands of the DNA. The DNA recognition sites for restriction enzymes are typically palindromic sequences 4 to 8 bases in length and the double stranded breaks occur within or adjacent to the recognition sites. For example, the restriction enzyme EcoRI recognizes and binds to the six nucleotide sequence of 5′-GAATTC-3′. In this case, the enzyme nicks the phosphodiester bond between the G and the A on both strands, generating two DNA fragments with so-called “sticky ends” (Table 1). Because a six base pair sequence such as GAATTC is encountered frequently in the human genome, treatment of genomic DNA with the EcoRI enzyme generates roughly one million DNA fragments. A single base change in a restriction enzyme recognition site prevents an endonuclease from binding to the DNA, resulting in no cleavage of the DNA. Conversely, a single base change may create a new recognition site where none existed prior to the base change. Different individuals in the population harbor a variety of benign point mutations that alter restriction enzyme recognition sites, thus resulting in a different collection of fragments. Such variation in the sizes of DNA fragments obtained by restriction endonuclease digestion is visualized by gel electrophoresis and Southern blotting; this procedure is termed “restriction fragment length polymorphism,” or RFLP, analysis (Figure 1). For gel electrophoresis, DNA fragments are loaded onto a porous, gelatinous material to which an electrical field is applied. By virtue of the inherent negative charge of DNA molecules, fragments migrate to the positive pole. Smaller fragments migrate faster than larger fragments thereby resolving in the gel on the basis of size. The fragments are visualized by the addition of a dye molecule, such as ethidium bromide, that intercalates between the DNA base pairs and fluoresces upon illumination with UV light. Other fluorescent dyes with greater sensitivity to 1 staining small quantities of DNA and less toxicity are also used. Often however, a particular fragment of diagnostic value is of interest and it is impossible to identify an individual fragment with many fragments resolved in the gel. The ability to visualize single DNA fragments requires the addition of a DNA probe in these situations that hybridizes specifically to the fragment of interest. A DNA probe is typically single stranded and complimentary to the gene or region of DNA interest. It is labeled with a fluorescent or radioactive tag so that the probe:target complex can be detected. Probes can be DNA fragments purified from restriction endonucleases digestion or small, synthetic DNA oligonucleotides are synthesized. Table 1. Examples of Commonly Used Restriction Endonucleases. Arrows show restriction sites of sequence. Type of Product Recognition Sequence Blunt ends AGCT TCGA 5’ AG….……………..CT 3’ 3’ TC………...………GA5’ EcoRI Escherichia coli strain R 5’ overhang GAATTC CTTAAG 5’ G…………….AATTC 3’ 3’CTTAA….............… G 5’ PstI Providencia stuatii 3’overhang CTGCAG GACGTC 5’CTGCA.....................G 3’ 3’G……….……ACGTC 5’ MnlI Moraxella nonliquefaciens Nonpalindromic sequence Enzyme Source AluI Arthrobacter luteus Fragment Ends CCTCNNNNNNN 5’CCTC(N7)3’ GGAGNNNNNNN 3’GGAG(N6)...N5’ A gene-specific probe is not hybridized to its complement in an agarose gel due to the difficulty of working with the gel matrix itself and due to any radioactive background that could occur with this type of labeling. Instead, the fragmented DNA is transferred to a dry filter and the probe is added. This is the essence of the Southern blot procedure (see Figure 1). Specifically, following gel electrophoresis, the DNA strands are denatured with a strong base while still in the gel. The single-stranded DNA is then transferred to a nylon or nitrocellulose membrane support by capillary action; sometimes application of a vacuum facilitates transfer. At this point, the denatured DNA is “fixed” to the membrane and a probe is applied under conditions that favor the re-establishment of DNA duplexes. As suggested above, it is the Watson-Crick hydrogen bond 2 base pairing between the single strand probe and its single-stranded complement restriction fragment that enables the probe to specifically anneal only to its complement. Visualization of a Figure 1. Southern Blotting technique is used to visualize fragments of variable lengths generated by various restriction endonucleases (RFLP analysis). Genomic DNA Restriction Endonuclease Fragmented Genomic DNA Large fragments - Small fragments + DNA separated on an agarose gel Transfer (blot) DNA to filter Hybridize (probe) filter with radioactive or non-radioactive probe to gene or region of interest; remove unbound probe Autoradiography or Development Visualization of fragments complementary to probe DNA fragment of interest is performed by laser-facilitated detection of the fluorescent probe or by exposure of the filter to X-ray film in the case of a radioactive probe. Thus, by using a 3 combination of restriction endonuclease digestion, gel electrophoresis, and Southern blotting, visualizing a DNA fragment of interest is accomplished. Southern blotting, as described, is a useful tool for identifying DNAs. Another similar tool is the Northern blot which has many similarities to the Southern blotting. The primary difference is that Northern blot provide visualization of RNAs and is therefore quite useful to determine the presence of RNAs, particularly mRNA, being expressed. The presence of mRNAs documents that the gene is indeed expressed which also validates the presence of a functional promoter and suggests the size of the protein. mRNAs that are smaller or larger than the expected size provide information suggesting a deletion or insertion in the gene or abnormal splicing. If the quantity of mRNA present is less than expected, this may suggest a weak promoter, which can occur for several reasons, or provoke questions about the half-life of the RNA. The Polymerase Chain Reaction (PCR) The introduction of polymerase chain reaction (PCR) has revolutionized DNA-based diagnostics. The rapid, inexpensive amplification of specific DNA sequences made possible with PCR has tremendously enabled both preparative and analytical procedures. PCR is the in vitro enzymatic amplification of a short (up to 5-6 kb) and specific DNA sequence. Amplification can be initiated with even a single DNA molecule and produces millions of copies in a period of a few hours. There are four essential components to PCR: two deoxyoligonucleotide primers, a thermostable DNA polymerase, target DNA, and nucleotides. The primers add specificity to the amplification by defining the flanking regions of DNA sequences to be amplified. Primers are “designed” to reflect the complimentary nucleotide sequence of the target. These are usually 15 – 30 nucleotides in length and synthesized by an automated process. Primers bind to targets in a 5’-3’ direction on each strand and amplification occurs between them. Amplification occurs during a series of denaturing, heating, and cooling phases that are repeated numerous times. These serve to dissociate double stranded DNA, allow primers to anneal, and facilitate new strand extension. The polymerase used in this procedure must be thermostable at high temperatures, a feature not characteristic of enzymes. The best known of the special thermostable polymerases was isolated from Thermus aquaticus. Designated DNA taq polymerase, it withstands repeated cycles of 95˚C or greater. During “denaturation,” the target DNA in the reaction is heated to 95oC rendering the target DNA single-stranded by breaking the hydrogen bonds between the two strands. The reaction is then cooled, typically to 45 – 65oC, to permit “annealing” of the single-stranded primers to complimentary sequences in the target DNA. Finally, the temperature is increased to 70-75oC to allow extension or synthesis of the new DNA most efficiently. During the extension phase, DNA polymerase uses the free 3′-OH group on the primers to synthesize new DNA. This 3 step or phase cycle is repeated with the newly synthesized DNA strands serving as templates for additional strand formation. Early in the process, the original genomic DNA is diluted out and the ends of the newly synthesized DNA strands are entirely defined by the primers. Overall, this cycle is repeated 20-35 times and the number of DNA copies doubles with each cycle, thus resulting in millions of copies of specific, primer-directed DNA fragments (see Karp Figure 18.48). It should be noted that the thermostable DNA polymerase, an excess of primers, and the 4 formation of newly synthesized DNA fragments that serve as templates for subsequent cycles permit the iterative cycles of amplification. Application of the PCR in molecular genetic diagnostics will be described below for detecting mutations. It should be apparent, however, that PCR has two direct utilities. First, it can provide abundant substrate for mutation detection strategies. Second, it can be a stand-alone diagnostic tool for detecting changes in DNA length, such as small insertions/deletions. In either case, PCR has greatly facilitated the era of genetic medicine by providing a rapid and inexpensive tool to biomedical scientists working on both diagnostic and research procedures (Table 2). Table 2. Comparison of PCR and RFLP RFLP Time Requirement Amount of DNA required Cost Sensitivity Days Micrograms to milligrams May be high with radioactivity usage and disposal; less costly if using non-radioactive probes Less sensitive to small quantities of DNA PCR Hours Picograms or less Rarely needs radioactivity and therefore less expensive Most sensitive to small quantities or copy number DNA Sequence Analysis In the post-Human Genome Project era, “unknown” regions of the chromosome are no longer sequenced to find new disease-causing mutations. More typically, genes or certain regions of genes are sequenced to detect a disease-specific mutation. Because the entire sequence of the human genome is known, PCR primers can be designed to amplify specific DNA for sequencing. Since DNA sequencing is greatly facilitated by abundant target DNA, only one application of PCR amplification of a specific DNA substrate is generally needed for sequencing. The most common method of DNA sequencing is the Sanger dideoxynucleotide chain terminating technique (Figure 2A). Here, heat denatured, single-stranded template, such as PCR products, is added to each of four tubes. Each tube contains a mixture of the four nucleotides (A, G, C, and T) that acts as a substrate for DNA polymerase. Each tube also contains one of the four chain terminating dideoxy ribonucleotides (ddA, ddG, ddC, or ddT). Hence the “A” tube contains have a mixture of the four normal radioactively labeled nucleotides (dA, dG, dT, and dC) plus the chain terminating ddA; a similar mixture of dNTPs to ddNTPs is found in the “G,” “C,” and “T” tubes. The incorporation of any dideoxy nucleotides prohibits further DNA polymerization because these lack the 3′-OH group required by DNA polymerase to add the next nucleotide. The addition of DNA polymerase and oligonucleotide primers, which are required to anneal to the target and initiate DNA polymerization via the free 3′-OH, to each reaction tube initiates DNA synthesis. However, the chains are terminated at different positions due to the 5 random insertion of a dideoxynucleotide instead of a normal deoxynucleotide. Stated differently, when a ddG is inserted by the DNA polymerase instead of a dG, synthesis stops at that point. In the “G” tube reaction, for example, some chains will be terminated at the initial G encountered by the DNA polymerase, while other chains will be halted at other G positions of the chain. In the “G” tube, the ratio of dGTP/ddGTP is such that there will be a number of chains that have terminated at each G position along the template. Figure 2. DNA sequencing. A. Sequencing done with a radioactive labeled deoxynucleotides. [used with permission: genetics.nbii.gov/ basic2.html. Probes may be radioactive or non-radioactive. B. Automated sequencing can be done with fluorescent dyes attached to dideoxynucleotides. A B 6 The four reactions are separated by gel electorphoresis on a polyacrylamide gel to produce a visual “ladder” of DNA fragments. Labeling either the DNA sequencing primers or the individual nucleotides with a radioactive or fluorescent tag enables this visualization. In automated sequencing, the ddNTPs are labeled with fluorescent dyes that are detected by a scanner. Because the gel lane containing “A” (from the “A” tube), “T” and so forth is known, it is a straightforward matter to read the sequencing ladder. The majority of DNA sequencing was once done by polyacrylamide gel electrophoresis but more recently is being replaced by capillary electrophoresis (Figure 2B). This technique adds automation to the process while adding several highly regarded features. DNA separation occurs in a capillary with a diameter of 25-100 μm, which provides a high ratio of surface area to volume that acts to dissipate heat produced. This feature allows the use of higher electric fields that decreases time of separation and increases resolution of DNA. Mutation Detection The true basis of molecular diagnostics is the detection of specific disease-causing mutations. Here, the most common methods associated genetic variation that are employed in laboratories for the detection of common diseases are discussed and include point mutations, deletions, and trinucleotide repeat expansions. Point Mutation Detection Mutation-specific RFLPs. In some single gene disorders, the causal mutation is a point mutation that alters a restriction endonuclease recognition site. This type of mutation can either abolish an existing recognition site or create a novel site. When this happens, it permits a mutation-specific test that can be used for diagnostic purposes. Perhaps the best example of a mutation-specific RFLP test is found in sickle cell anemia. Sickle cell anemia results from an A to T missense mutation and the substitution of a valine for a glutamate at the sixth amino acid of the β-globin polypeptide. This base change affects the 5′-CCTNAGG-3′ (where N can be any nucleotide) recognition site for the MstII restriction endonuclease because the central A is replaced by a T, rendering a similar but different 5-CCTNTGG-3′ sequence. MstII does not recognize or cleave this altered DNA sequence. Hence, sickle cell anemia patients differ from the normal population by the loss of this particular restriction site, resulting in a RFLP for sickle cell anemia. In the laboratory, this is recognized with agarose gel electrophoresis where normal individuals have two smaller DNA fragments that corresponded to the affected individual’s single, longer DNA fragment (Figure 3). While specific and sensitive, this methodology is relatively rare in practice because the majority of point mutations do not alter a restriction endonucleases recognition site. As suggested earlier, PCR can also be used to amplify a fragment of interest for restriction analysis. The fragment sizes may differ between genomic 7 DNA versus PCR-generated DNA and a probe may be required to visualize a fragment in genomic DNA, but the results still demonstrate the affect of the mutation. Figure 3. RFLP analysis of the -globin gene and sickle cell anemia. A. An A-to-T point mutation occurs in codon 6. B. The mutation eliminates a recognition site for MstII. Individuals with sickle trait are heterozygous and have a 1.15 kb and 1.35 kb fragments. Individuals with sickle cell anemia have 2 longer 1.35 kb fragments. A B C Allele specific oligonucleotides. Thousands of single base changes are associated with human disease. This has necessitated the development of other methods to identify individual point mutations because mutation-specific RFLPs are relatively rare. One of these methods is 8 allele specific oligonucleotide (ASO) hybridization (Figure 4). For this, a DNA synthesizer creates short, synthetic oligonucleotides that are an exact compliment of the normal DNA sequence. These, in turn, are radioactively or fluorescently labeled and used to probe patient and/or control DNA in a process similar to the Southern blot probing. During the hybridization process, these short oligonucleotides only bind to a perfect sequence complement and not to any sequence with even a single mismatched base. Thus, the oligonucleotides only bind normal DNAs, making it simple to distinguish between normal DNA and mutant DNA. Conversely, a short oligonucleotide may also be synthesized that is a perfect complement to a mutant DNA sequence. For example, an ASO can be synthesized that hybridizes only to the portion of the globin gene that contains the sickle cell anemia mutation. Such an oligonucleotide does not bind to normal DNA because it is mismatched at the base altered in sickle cell anemia. A mutationspecific ASO, then, can be used to screen patients, family members, and even members of the general population for the presence of the sickle cell anemia mutation. Figure 4. Allele specific oligonucleotides can be designed to identify specific mutations either known to occur in a family or that occur at a high frequency in the population. In this example, if the ASO are designed to the normal and mutant region of the -globin gene, the fetus is a normal carrier rather than affected. Single-stranded DNA samples from family members Single-stranded DNA is made from a normal allele (A) GGACTCCTC Single-stranded DNA is made from a mutant allele (a) GGACTACTC A probe is made for each allele CCTGAGGAG CCTGATGAG Probes are hybridized to sample DNAs Father Child Mother Fetus Probe for normal allele (A) Probe for mutant allele (a) Deduced Genotypes Aa Aa AA Aa 9 DNA Sequencing In special cases where a patient is clearly suffering from a particular disease but does not harbor known genetic variants associated with the disease, the candidate gene may be subjected to complete or partial automated DNA sequence analysis (Figure 2). DNA sequencing is much more labor intensive and time consuming if the specific mutation is unknown, but it is an excellent approach for the discovery of new or rare mutations. However, this technique is so valuable that increased technological developments are providing improvements designed to reduce the time and cost of sequencing; it is not unthinkable that samples currently referred to a research lab for evaluation will be sequenced in a diagnostic lab in the near future. Deletion Detection Very small deletions, typically single base deletions that cause disease may be screened for using the same methodologies as employed for point mutations. Larger deletions, however, require a different approach. PCR is ideal for detecting deletions in the 100 base pair to 4 kb range since primer pairs amplify both a normal and mutated allele containing a deletion. Following PCR, DNA fragments are separated by electrophoresis and compared to molecular weight standards. Deleted alleles are obviously not as large as the non-deleted forms and hence easily visualized. Trinucleotide Repeat Expansion Detection Expansions of trinucleotide sequences are a relatively common genetic mechanism for neurological disease. Fragile X syndrome, Huntington disease, myotonic dystrophy, spinobulbar muscular atrophy and Friedreich’s ataxia are all examples of disease caused by expanding trinucleotide repeats. This class of mutation poses a special challenge for diagnostics as illustrated by the Fragile X syndrome. Fragile X1 is due to an expansion of a CGG trinucleotide in the 5′ untranslated region of the FMR1 gene. Three classes of FMR1-associated CGG expansions are recognized in the population: normal chromosomes contain between 5 and 50 repeats; premutation chromosomes contain between 50 and 200 repeats, and full mutation chromosomes (affected individuals) feature 200 to 2000 CGG repeats. Distinguishing between these three allelic repeats seems, intuitively, an ideal task for PCR. In practice however, this is true for normal and premutation alleles; PCR primers flank the site of the CGG expansion and amplify the alleles. The same is not always true for the full mutation because full mutation expansions can reach 6 kb in length, which exceeds the size where accurate genotyping can be done on the basis of PCR alone. Thus, Southern blotting is typically done when Fragile X syndrome is suspected. In this case, a probe is used that hybridizes proximal to the expanded region. In this way, very large expansions can be detected. The dividing line between using PCR and Southern to size the repeat tract is between 70-100 repeats. Southern blots cannot identify a precise repeat size at the normal and low permutation range, but PCR is unable to correctly identify repeat tracts over 70-100 units. It is important to note that DNA diagnostics pervades the entirety of the health care system today. A large number of laboratories utilizing genetic techniques now exist and stand as a testament to the ubiquity of genetic defects in medicine. Even in rural or remote settings, the physician sends blood samples to a laboratory for DNA analysis. The association between 1 Fragile X syndrome will be discussed in detail for Case 8. 10 medicine and molecular genetics will grow more comprehensive as additional insights are made regarding genetic predisposition to disease and the role of genetics in pharmaceutical efficacy. 11