Molecular Basis of Inheritance Notes PDF

Molecular Basis of Inheritance DNA: The Genetic material Φ×174 bacteriophage - 5386 nucleotides λ bacteriophage - 48502 base pairs (bp) Escherichia coli - 4.6 × 106 bp Haploid content of human DNA - 3.3 × 109 bp Structure of the polynucleotide chain Three components of...

Molecular Basis of Inheritance DNA: The Genetic material Φ×174 bacteriophage - 5386 nucleotides λ bacteriophage - 48502 base pairs (bp) Escherichia coli - 4.6 × 106 bp Haploid content of human DNA - 3.3 × 109 bp Structure of the polynucleotide chain Three components of a polynucleotide chain: 1. A nitrogenous base 2. A pentose sugar (ribose for RNA, deoxyribose for DNA) 3. A phosphate group Two types of nitrogenous bases: 1. Purines - Adenine and Guanine 2. Pyrimidines - Cytosine, Thymine (in DNA), and Uracil (in RNA) An N-glycosidic linkage links a nitrogenous base to the OH of 1’C pentose sugar to form nucleoside, such as 1. Adenosine (RNA) or deoxyadenosine (DNA) 2. Guanosine (RNA) or deoxyguanosine (DNA) 3. Cytidine (RNA) or deoxycytidine (DNA) 4. Uridine (RNA) or deoxythymidine (DNA) Phosphate group linked to OH of 5’C of a nucleoside through phosphoester linkage Two nucleotides are linked through 3’-5’ phosphodiester linkage to form a dinucleotide. Thymine - 5-methyl uracil Figure 1: The difference in the structures of Uracil and Thymine 5’ end of polynucleotide chain - free phosphate moiety  3’ end of polynucleotide chain - free OH group In RNA, every nucleotide residue has an additional -OH group present at the 2’ position in the ribose. Figure 2: Basic structure of a double-stranded DNA History of DNA DNA as an acidic substance present in the nucleus - first identified by Friedrich Meischer in 1869. He named it ‘Nuclein.’ Double Helix Model of DNA structure In 1953: James Watson and Francis Crick proposed based on the X-ray diffraction data from Maurice Wilkins and Rosalind Franklin. The characteristic feature of the model was base pairing between the two strands of polynucleotide chains (a unique part of the model). Chargaff’s Rule: 1. Adenine:Thymine = 1:1 2. Guanine:Cytosine = 1:1 3. Adenine+Guanine = Thymine+Cytosine Salient features of the double helix DNA: 1. Backbone made of sugar-phosphate 2. Antiparallel polarity 3. The bases in the two strands are paired through hydrogen bonds (H-bonds), forming base pairs (bp) 4. Coiled in a right-handed fashion 5. Pitch of the helix: 3.4 nm 6. Distance between a base pair in a helix: 0.34 nm Figure 3: Diagram showing the double helix model of DNA proposed by Watson and Crick Packaging of DNA Helix In prokaryotes: 1. DNA is not scattered throughout the cell. 2. Negatively charged DNA is held with positively charged proteins in a region termed ‘nucleoid.’ In eukaryotes: 1. Positively charged, basic proteins - histones 2. The basic nature of histones means a high concentration of lysine and arginine AAs (basic AAs) 3. Eight histones form together a histone octamer 4. DNA strand + histone octamer = ‘Nucleosome’ 5. One nucleosome - 200 bp DNA strand Figure 4: Nucleosomes are an essential element involved in DNA Packaging in Eukaryotes Chromatin: 1. Nucleosomes constitute the repeating unit of a structure in the nucleus called chromatin. 2. Thread-like stained (colored) bodies are seen in the nucleus 3. The nucleosomes in chromatin are seen as ‘beads-on-string’ structures when viewed under an electron microscope 4. The packaging of chromatin at a higher level requires an additional set of proteins that collectively are referred to as Non-histone chromosomal (NHC) proteins. 5. Euchromatin: Loosely packed, lightly stained, transcriptionally active 6. Heterochromatin: Densely packed, dark stained, transcriptionally inactive The Search for Genetic Material: Scientific Inquiry T.H Morgan’s Group showed that genes exist as parts of chromosomes. DNA and Protein were leading candidates for genetic material. Until the 1940s, the case for proteins seemed stronger, because 1. Biochemists identified proteins as a class of biomacromolecules with significant heterogeneity and specificity of function-essential requirements for hereditary material. 2. Little was known about nucleic acids- physical and chemical properties seemed too uniform. This view was changed gradually as the role of heredity was worked out in studies of bacteria and viruses that infect them. Transforming Principle: 1. 1928-British Medical Officer- Frederick Griffith 2. Griffith- trying to develop a vaccine against pneumonia 3. Streptococcus pneumoniae- two strains: a. Pathogenic(disease-causing) b. Non-pathogenic(harmless) 4. Pathogenic(dead) bacteria + Non-pathogenic(live) bacteria ⇒ Pathogenic(live) bacteria 5. This trait was acquired by all the descendants of the transformed bacteria 6. Griffith called this phenomenon transformation 7. Oswald Avery, Maclyn McCarty, and Colin MacLeod identified the transforming substance as DNA. 8. Continued doubt about DNA as genetic material- Little was known about DNA. In 1952- Alfred Hershey & Martha Chase: 1. Showed that DNA is the genetic material of a phage T2 2. T2 phage infects Escherichia coli(E. coli) 3. Used radioactive isotope of sulfur for protein and a radioactive isotope of phosphorus for DNA 4. Radioactive sulfur atoms were incorporated only into the protein of the phage, but radioactive phosphorus entered the E. coli cell in the form of DNA. 5. Landmark study because it provided robust evidence that nucleic acids, rather than proteins, are the hereditary material, at least for specific viruses.  Figure 5: The Hershey - Chase Experiment Properties of Genetic Material (DNA versus RNA) The Hershey-Chase experiment unequivocally resolved the debate between proteins and DNA as genetic material. Subsequently, it became clear that RNA is the genetic material in some viruses, e.g., Tobacco Mosaic Virus and QB bacteriophage. What makes DNA and RNA molecules so unique that they can function as genetic material? They must fulfill the following criteria: 1. Generating replica possible 2. Chemically and structurally stable 3. There should be scope for slow changes (mutation) that are required for evolution 4. Able to express itself in the form of ‘Mendelian Characters’ Why is DNA a better genetic material for storing genetic information than RNA? 1. The two strands of DNA being complementary if separated, by heating come together when appropriate conditions are provided 2. 2’-OH group present at every nucleotide in RNA is a reactive group and makes RNA labile and easily degradable  3. RNA is also known to be catalytic, hence reactive. DNA chemically is less reactive and structurally more stable as compared to RNA 4. The presence of thymine at the place of uracil also confers additional stability to the DNA Why is RNA a better genetic material for transmitting genetic information than DNA? 1. RNA being unstable mutates at a faster rate\ 2. RNA can directly code for the synthesis of proteins and hence can easily express the characters. DNA is, however, dependent on RNA for the synthesis of proteins 3. The protein synthesizing machinery has evolved around RNA Figure 6: Picture showing structural differences between the two molecules RNA: The First Genetic Material Enough evidence to suggest that essential life processes evolved around RNA. RNA used to act as a genetic material as well as a catalyst. DNA has evolved from RNA with chemical modifications that make it more stable Semiconservative DNA Replication It has been proved that DNA replicates semiconservatively. It was first shown in Escherichia coli and subsequently in higher organisms, such as plants and human cells  Meselson and Stahl’s experiment (1958): 1. They grew E. coli in a medium containing 15NH4Cl (15N is the heavy isotope of nitrogen) as the only nitrogen source for several generations 2. Then they transferred the cells into a medium with normal 14NH4Cl, took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained double-stranded helices. 3. The DNA extracted from the culture one generation after the transfer from 15N to 14 N medium [that is, after 20 minutes; E. coli divides in 20 minutes] had a hybrid or intermediate density. DNA extracted from the culture after another generation [that is 40 minutes, II generation] was composed of equal amounts of this hybrid DNA and ‘light’ DNA Taylor ⇒ performed a similar experiment on Vicia faba in 1958 and proved that DNA in chromosomes also replicates semiconservatively Figure 7: Image illustrating the Meselson - Stahl Experiment Other types of Replication Conservative model: The two parental strands somehow come back together after the process Dispersive model: All four strands of DNA following replication have a mixture of old and new DNA Figure 8: Models of DNA Replication The Machinery for DNA Replication DNA-dependent-DNA Polymerase: 1. The main enzyme - uses a DNA template to catalyze the polymerization of deoxynucleotides 2. Highly efficient, as they have to catalyze the polymerization of a large number of nucleotides in a concise amount of time 3. E. coli that has only 4.6×106 bp (compare it with a human whose diploid content is 6.6×109 bp), completes the process of replication within 18 minutes; that means the average rate of polymerization has to be 2000 bp per second Energetically, replication is a costly process. Deoxyribonucleotide phosphates serve dual purposes: 1. Act as substrates 2. Provide energy for polymerization reaction (the two terminal phosphates are high-energy phosphates, same as in the case of ATP 3. Replication fork: 1. For long DNA molecules, since the two strands of DNA cannot be separated in their entire length (due to very high energy requirement), the replication occurs within a small opening of the DNA helix, referred to as the replication fork 2. The DNA-dependent DNA Polymerases catalyze polymerization only in one direction, that is 5’→3.’ 3. On one strand (the template with polarity 3’→5’), the replication is continuous, while on the other (the template with polarity 5’→3’), it is discontinuous. Figure 9: The structure of the DNA Replication Fork The enzyme DNA ligase later joins the discontinuously synthesized fragments. Replication of chromosomal DNA begins at particular sites called origins of replication. E. coli chromosome is circular and has a single origin Prokaryotic DNA Replication ( as in E. coli): Proteins that initiate DNA replication recognize origin sequence and attach to DNA. The two strands get separated. The separation strands form replication ‘bubbles.’ Replication proceeds in both directions until the entire molecule gets copied. Figure 10: Diagram illustration of Replication ‘bubble’ in Prokaryotic DNA Replication Eukaryotic DNA Replication: It may have hundreds or even a few thousand replication origins. Multiple replication bubbles form and eventually fuse, thus speeding up the copying of the very long DNA molecules.  Figure 11: Diagram illustration of Replication ‘bubbles’ in Eukaryotic DNA Replication At the end of each replication bubble is a replication fork. Helicases are the enzymes that untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands. After the parental strands separate, single-stranded binding proteins bind to the unpaired DNA strands, keeping them from repairing. Topoisomerase helps relieve the strain caused by untwisting of the double helix by breaking, swiveling, and rejoining DNA strands. Transcription The process of copying genetic information from one strand of DNA into RNA is called transcription. The principle of complementarity governs the transcription process, except the adenosine complements now form base pairs with uracil instead of thymine. Unlike replication, the DNA of an organism gets duplicated; in transcription, only a segment of DNA and only one of the strands is copied into RNA. Why are both strands not copied during transcription? 1. If both strands act as a template, they will code for RNA molecules with different sequences (Remember, complementarity does not mean identical)  2. The two RNA molecules, if produced simultaneously, would be complementary to each other, hence would form a double-stranded RNA. Transcription Unit A transcription unit in DNA is defined primarily by the three regions in the DNA: 1. A Promoter 2. The Structural Gene 3. A Terminator 4. Template strand: The strand with polarity 3’→5’ acts as a template, thus referred to as the template strand Coding strand: The coding strand is the other strand with polarity 5’→3’ and the same sequence as RNA (except the thymine in place of uracil). Although referred to as the coding strand, it does not code for anything. Promoter- the DNA sequence where RNA polymerase attaches and initiates transcription Terminator- the sequence that signals the end of transcription Transcription factors: 1. Present in eukaryotes 2. Collection of proteins 3. Help guide the binding of RNA polymerase 4. Initiation of transcription Transcription initiation complex- Whole complex of transcription factors and RNA polymerase II bound to the promoter Cistron: A segment of DNA coding for a polypeptide The structural gene in a transcription unit could be said as monocistronic (mostly in eukaryotes) or polycistronic (mostly in bacteria or prokaryotes) Exons: The coding sequences or expressed sequences Introns: Non-coding sequences which interrupt exons; do not appear in mature or processed RNA Spliceosome: 1. Large complex made of proteins and small RNAs 2. Function- removal of introns 3. Binds to several short nucleotide sequences along an intron, including key sequences at each end Exon shuffling- the presence of introns in a gene may facilitate the evolution of new and potentially beneficial proteins due to this process.  Figure 12: Coding and Template strand The Transcription Process Initiation: 1. RNA polymerase binds to the promoter and initiates transcription. 2. It uses nucleotide triphosphates as substrate and polymerizes in a template-dependent fashion following the rule of complementarity Elongation: 1. Initiation facilitates the opening of the helix and continues elongation 2. Transcription progresses at a rate of about 40 nucleotides per second in eukaryotes Termination: 1. In bacteria: - Transcription proceeds through a terminator sequence in the DNA - The transcribed terminator (an RNA sequence) functions as the termination signal - DNA polymerase detaches from the DNA and releases the transcript 2. In eukaryotes: RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal sequence, which specifies a polyadenylation signal in the pre-mRNA RNA Polymerase is only capable of catalyzing the process of elongation. It associates transiently with the initiation factor (σ) and termination factor (ρ) to initiate and terminate the transcription.In bacteria, since the mRNA does not require any processing to become active, and since transcription and translation take place in the same compartment (there is no separation of cytosol and nucleus in bacteria), the translation can often begin much before the mRNA is fully transcribed. As a consequence, transcription, and translation can be coupled in bacteria. Two additional complexities in eukaryotes: 1. There are at least three RNA polymerases in the nucleus (in addition to the RNA polymerase found in the organelles). There is a clear-cut division of labor. RNA polymerase I transcribe rRNAs (28S, 18S, and 5.8S), whereas RNA polymerase III is responsible for transcribing tRNA, 5srRNA, and snRNAs (small nuclear  RNAs). RNA polymerase II transcribes the precursor of mRNA, the heterogeneous nuclear RNA (hnRNA). 2. Primary transcripts contain both the exons and the introns and are non-functional. Hence, it is subjected to splicing (removal of introns and joining exons in defined order). hnRNA undergoes capping and tailing, after which it is called mRNA. 3. Capping: an unusual nucleotide (methyl guanosine triphosphate) is added to the 5’-end of hnRNA Tailing: adenylate residues (200-300) are added at the 3’-end in a template-independent manner Figure 13: Stages of transcription - explained. RNA Processing RNA Processing in the nucleus and the export of mature RNA to the cytoplasm provide opportunities for regulating gene expression not available in prokaryotes Alternative RNA splicing: Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Alternative RNA splicing can significantly expand the repertoire of a eukaryotic genome. Proposed as one explanation for the surprisingly low number of human genes counted when the human genome was sequenced. More than 90% of human protein-coding genes likely undergo alternative splicing. The extent of alternative splicing dramatically multiplies the number of possible human proteins, which may correlate better with the complexity of form than the number of genes. Figure 14: Alternative RNA Splicing Genetic Code George Gamow(physicist) argued that since there are only four bases and if they have to code for 20 amino acids, the code should constitute a combination of bases. He suggested that to code for all the 20 amino acids, the code should be made up of three nucleotides. A very bold proposition since a permutation combination of 43 (4×4×4) would generate 64 codons, generating many more codons than required. The chemical method developed by Hargobind Khorana was instrumental in synthesizing RNA molecules with defined combinations of bases (homopolymers and copolymers). Marshall Nirenberg’s cell-free system for protein synthesis finally helped the code to be deciphered. Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerizing RNA with defined sequences in a template-independent manner (enzymatic synthesis of RNA). The salient features of the genetic code: 1. The codon is a triplet; 61 codons code for amino acids, and three codons do not code for any amino acids; hence they function as stop codons 2. Some amino acids are coded by more than one codon; therefore, the code is degenerate 3. The codon is read in mRNA in a contiguous fashion; there are no punctuations 4. The code is nearly universal; for example, from bacteria to human UUU would code for Phenylalanine (phe). Some exceptions to this rule have been found in mitochondrial codons and some protozoa. 5. AUG has dual functions. It codes for Methionine (met) and acts as an initiator codon. 6. UAA, UAG, and UGA are stop terminator codons Figure 15: Codon triplet code Mutations and Genetic Code Point mutation is a change of single base pair in the gene. Insertion or deletion of one or two bases changes the reading frame from the point of insertion or deletion; such mutations are referred to as frameshift insertion or deletion mutations. tRNA - The Adapter Molecule Francis Crick postulated the presence of an adapter molecule that would, on the one hand, read the code and, on the other hand would, bind to specific amino acids. The tRNA then called sRNA (soluble RNA), was known before the genetic code was postulated. Each tRNA molecule enables the translation of a given mRNA codon into a specific amino acid. tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long (compared to hundreds of molecules for most mRNA molecules). A tRNA molecule looks like a clover leaf. The tRNA molecule is a translator in the sense that, in the context of the ribosome, it can read a nucleic acid word (the mRNA codon) and interpret it as a protein word (the amino acid). In a eukaryotic cell, tRNA, like mRNA, is made in the nucleus and then travels to the cytoplasm, where it will participate in the process of translation. Instances of molecular recognition required for the accurate translation of a genetic message. A tRNA that binds to an mRNA codon specifying a particular amino acid must carry that amino acid, and no other, to the ribosome. The pairing of the tRNA anticodon with the appropriate mRNA codon. Figure 16: Structure of tRNA Translation It refers to the polymerization of amino acids to form a polypeptide. The order and sequence of amino acids are defined by the sequence of bases in the mRNA. In the first phase, amino acids are activated in the presence of ATP and linked to their cognate tRNA - a process called charging of tRNA or aminoacylation of tRNA. When the small subunit of the ribosome encounters an mRNA, the process of translation of the mRNA to protein begins. The ribosome also acts as a catalyst (23S rRNA in bacteria is the enzyme - ribozyme) for the formation of peptide bond. An mRNA also has some additional sequences that are not translated and are referred to as untranslated regions (UTRs). The UTRs are present at 5’-end (before the start codon) and 3’-end (after the stop codon). They are required for an efficient translation process. In eukaryotes, the small subunit, with the initiator tRNA already bound, binds to the 5’ cap of the mRNA and then moves, or scans, downstream along the mRNA until it reaches the AUG start codon, where the initiator tRNA then hydrogen-bonds. Translation initiation complex = mRNA + initiator tRNA + small ribosomal subunit. Initiation factors- proteins that are required to bring the components of the translation initiation complex together. A polypeptide is always synthesized in one direction, from the initial methionine at the amino end, also called N-terminus, towards the final amino acid at the carboxyl end, also called C-terminus. Amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain. Elongation factors- proteins involved in each addition; occur in a three-step cycle. The elongation of polypeptide takes less than a tenth of a second in bacteria and is repeated as each amino acid is added until the polypeptide is completed. Release factor is a protein shaped like an aminoacyl tRNA that binds directly to the stop codon in the A site and causes the addition of a water molecule instead of an amino acid to the polypeptide chain. Breakdown of the translation assembly requires the hydrolysis of two or more GTP molecules. Figure 17: Translation Regulation of Gene Expression In eukaryotes, the regulation could be exerted at 1. Transcriptional level (formation of primary transcript) 2. Processing level (regulation of splicing) 3. Transport of mRNA from the nucleus to the cytoplasm 4. Translational level 5. It is the metabolic, physiological, or environmental conditions that regulate the expression of genes. The development and differentiation of embryos into adult organisms are also a result of the coordinated regulation of the expression of several sets of genes. In a transcription unit, the activity of RNA polymerase at a given promoter is regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulated proteins can act both passively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is, in many cases, regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons, and in most cases, the sequences of the operator bind a repressor protein. The Lac Operon The elucidation of the lac operon was also a result of a close association between a geneticist, Francois Jacob, and a biochemist, Jacques Monod. They were the first to elucidate a transcriptionally regulated system. Examples of operons: lac operon, trp operon, ara operon, his operon, val operon, etc. The lac operon consists of one regulatory gene (the i gene) and three structural genes (z, y, and a). Functions of the genes: 1. The i gene: codes for the repressor of the lac operon 2. The z gene: codes for β-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the disaccharide lactose into its monomeric subunits, galactose and glucose 3. The y gene: codes for permease, which increases the permeability of the cell to β-galactosides 4. The a gene: encodes a transacetylase. Lactose is the inducer since it is the substrate for the enzyme β-galactosidase and regulates switching on and off the operon. In the absence of a preferred carbon source such as glucose, if lactose is provided in the growth medium of the bacteria, the lactose is transported into the cells through the action of permease. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. Regulation of lac operon by a repressor is referred to as negative regulation. Figure 17: The lac operon Human Genome Project (HGP) Human Genome Project (HGP) was launched in 1990 with the establishment of genetic engineering techniques where it was possible to isolate and clone any piece of DNA and the availability of simple and fast techniques for determining DNA sequences. The human genome is said to have approximately 3 × 109 bp, and if the cost of sequencing required is US $3 per bp (the estimated cost in the beginning), the project's total cost would be approximately US $9 billion. If the obtained sequences were to be stored in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to keep the information of DNA sequence from a single human cell. HGP was closely associated with the rapid development of a new area in biology called Bioinformatics. Goals of HGP: 1. Identify all the approximately 20,000-25,000 genes in human DNA 2. Determine the sequences of the 3 billion chemical base pairs that make up human DNA 3. Store this information in databases 4. Improve tools for data analysis 5. Transfer related technologies to other sectors, such as industries 6. Address the ethical, legal, and social issues (ELSI) that may arise from the project Expressed Sequence Tags (ESTs): all the genes that are expressed as RNA. Sequence annotation: assigning different regions in the sequence with functions The commonly used hosts were bacteria and yeast, and the vectors were called BAC (bacterial artificial chromosomes), and YAC (yeast artificial chromosomes). The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger. Microsatellites are the repetitive DNA sequences. Salient Features of the Human Genome The human genome contains 3164.7 million nucleotide bases. The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases. The total number of genes is estimated at 30,000 - much lower than previous estimates of 80,000 to 140,000 genes. The functions are unknown for over 50 percent of the discovered genes. Less than 2 percent of the genome codes for proteins. Repeated sequences make up a substantial portion of the human genome. Chromosome 1 has the most genes (2968), and the Y has the fewest (231). Scientists have identified about 1.4 million locations where single-base DNA differences (SNPs - single nucleotide polymorphism) occur in humans. DNA Fingerprinting DNA Fingerprinting involves identifying differences in some specific regions in DNA sequence called repetitive DNA because, in these sequences, a small stretch of DNA is repeated many times. The bulk DNA forms a major peak, and the other small peaks are referred to as satellite DNA. Depending on the base composition, length of the segment, and the number of repetitive units, the satellite DNA is classified into many categories, such as micro-satellites, mini-satellites, etc. These sequences typically do not code for any proteins, but they form a large portion of the human genome. DNA Polymorphism, that is variation at the genetic level arises due to mutations, as a consequence of which an inheritable mutation is observed in a population at a high frequency. Alec Jeffreys initially developed the technique of DNA Fingerprinting. He used satellite DNA as a probe that shows a very high degree of polymorphism. It was called Variable Number of Tandem Repeats (VNTR). The technique involved Southern blot hybridization using radiolabeled VNTR as a probe. Steps of VNTR technique: 1. Isolation of DNA 2. Digestion of DNA by restriction endonucleases 3. Separation of DNA fragments by electrophoresis 4. Transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon 5. Hybridization using labeled VNTR probe 6. Detection of hybridized DNA fragments by autoradiography The VNTR belongs to a class of satellite DNA referred to as mini-satellite. In addition to application in forensic science, it has much broader applications, such as in determining population and genetic diversities. Currently, many different probes are used to generate DNA fingerprints. Using CRISPR to Edit Genes and Correct Disease-Causing Mutations Gene editing: 1. Altering genes in a specific, predictable way 2. Aim- to change particular genes in living cells, in part to try to correct genes that cause disease CRISPR-Cas9 System: 1. A powerful, new technique for gene editing 2. Genetic engineering- the direct manipulation of genes for practical purposes 3. Cas9- a bacterial protein that helps defend bacteria against the viruses that infect them  Cas9: 1. A nuclease that cuts double-stranded DNA molecules 2. Will cut any sequence to which it is directed 3. Directed to its target by a guide RNA molecule that it binds and uses as a homing device, cutting both strands of any DNA sequence that is complementary to the guide RNA 4. The guide RNA in the complex is engineered to be complementary to the “target” gene. 5. It cuts both strands of the target DNA, and the resulting broken ends of DNA trigger a DNA repair system. 6. This technique is a highly successful way for researchers to “knock out” (disable) a given gene to study what the gene does in an organism. CRISPR technology has the potential to treat or even cure human diseases that have a genetic basis, such as sickle cell anemia, Alzheimer’s, and Parkinson’s disease, as well as some types of cancer. Jennifer Doudna- a co-discoverer of CRISPR-Cas9- recognized its incredible potential and the danger of its misapplication.