BIO 141 LE 1-7-14 Molecular Genetic Analysis PDF

released, and the ribosome dissociates from the mRNA. IV. Control of Gene Expression Bacteria have evolved various mechanisms to regulate gene expression in response to intracellular or environmental conditions. This allows them to adapt to changes in their environment and optimize the use of their...

released, and the ribosome dissociates from the mRNA. IV. Control of Gene Expression Bacteria have evolved various mechanisms to regulate gene expression in response to intracellular or environmental conditions. This allows them to adapt to changes in their environment and optimize the use of their limited resources. One of the primary mechanisms of gene expression control in bacteria is transcriptional regulation. Bacteria use transcriptional regulators, such as repressors and activators, that can bind to specific DNA sequences (called operator or promoter regions) and modulate the rate of transcription. Repressors are proteins that bind to the operator region and block the binding of RNA polymerase, thereby inhibiting transcription. Activators, on the other hand, bind to the promoter region and facilitate the recruitment of RNA polymerase, increasing the rate of transcription. Bacteria also employ post-transcriptional and post-translational mechanisms to control gene expression. These include the use of small regulatory RNAs (sRNAs) that can bind to mRNA molecules and modulate their stability or translation, as well as the regulation of protein activity through mechanisms like phosphorylation or proteolysis. MOLECULAR GENETIC ANALYSIS I. Restriction Site Mapping & Molecular Analysis Restriction Enzymes - Isolated DNA can be cut at specific sites by enzymes called restriction enzymes Isolated from bacteria Recognize and cleave DNA at 4 to 8 specific nucleotides in a palindromic sequence ○ Sequence that reads the same 5’ to 3’ on one strand and 3’ to 5’ direction on complementary strand. If they cut symmetrically between two bases opposite each other on complementary strands → resulting DNA have blunt ends If the endonuclease makes staggered cuts across the two DNA strands leaving each fragment an overhand of unpaired bases → ends of DNA fragments are said to be sticky. - These ends can pair with single stranded overhangs of another piece of DNA cut by the same restriction enzyme. → Different DNA fragments with sequence-specific DNA sequences called a restriction site. Restriction Enzyme Digests - recognizes and cleaves DNA sequence called a restriction site. Background and Nomenclature Where do R.E. come from? Adaptation of bacteria that act as defense mechanism against viruses, known as bacteriophages. Due to addition of methyl groups, R.E. can only recognize and phage DNA R.E. names ○ HindIII, NotI, EcoRI, BamHI, TaqI, HistII ○ First three letters refer to the organism which it was ○ ○ isolated from (e.g. EcoRI; Eco → E.coli) Second letter, if necessary, refers to the bacterial strain from which it was isolated. Roman numeral indicates whether it was the first, second, or 3rd enzyme isolated from a particular organism Basic Principles RE recognizes a series of nucleotides usually 4 base pairs long called a recognition site. At specific nucleotides within the sequence, the enzyme will break the phosphodiester bonds in the DNA backbone Direct recognition sites are usually palindromic (sequence reads the same forwards and backwards) ○ When the palindrome is found a complementary strand of the DNA molecule it is called an inverted-repeat palindrome RE can leave different types of ends once the DNA is cleaved. ○ Sticky ends - leave 3’ and 5’ overhangs ○ Blunt ends - leave no overhangs. The type of end dictates how the DNA fragment is isolated by the KED; will be recombined with other DNA fragments in a process called ligation. Restriction Enzyme Digests Digestive reaction typically consists of ○ Deionized water ○ DNA to be cut ○ Buffers specific to the enzyme used ○ Protein called bovine serum albumin (BSA) → will stabilize the reaction by preventing enzymes from sticking to the sides of the container that houses the digest. R.E. BSA required Buffer Incubation Temp BamH1 Yes X 37 C NotI No Y 37 C SpeI Yes Z 50 C May have different buffers, incubation temperatures, and requirements for BSA. 1. Retrieve the restriction enzyme from the fridge. Keep restriction enzymes on ice or thermoresistant containers to make sure there is optimal activity for future reactions. 2. Microfuge tubes: a volume of sterile nuclease free water that will yield a final reaction volume of 20 microliters. 10x restriction buffer, BSAM 1 microgram of DNA, 2-10 units of enzyme. 1 unit = 1 microgram DNA digested in 1 hr of 37C in 50 microliter. 3. Mix by vortexing, centrifuge briefly at 12000 times g in a microcentrifuge. 4. Incubate at an optimal temperature for restriction enzyme: 37C in a heating block for 1-4 hours. 5. Incubate the reaction mixture at 65 C to heat and activate restriction enzymes. 6. Prolonged incubation times may result in star activity: cutting at sites that are similar but distinct from typical digestion sites. 7. Following inactivation, DNA should be run in an agarose gel to ensure that the digest was successful. → Sometimes multiple enzymes have to be used to generate a specific DNA fragment. Check if buffer conditions and incubation temperatures are compatible between two enzymes. If so, double digest may be performed. pieces and then identifying the locations of the breakpoints. → DNA segment that shows the positions of the restriction sites is called the restriction map. Samples: → Use enzymes sequentially if conditions are not compatible. Purify DNA following the initial digest, then do a second digest. → The use of control DNA with known restriction sites allows the enzyme activity to be tested. Applications: 1. Screening Samples: can be used diagnostically to identify samples. → Missing 6kb → Restriction Fragment Length Polymorphisms (RFLPs) → loading digests into specialized chips (placed into a machine called bioanalyzer) - Researchers can examine DNA fragment sizes produced by the digests to determine the authenticity of the sample → Different banding patterns of the same gene from a given species called restriction fragment length polymorphism (RFLPs) 2. Subcloning – isolate a fragment of DNA in one plasmid and insert it into another so the desired fragment can be replicated using bacteria. 3. Detecting SNPs - by employing PCR to introduce genes to very specific locations → Can be used to Determine the presence of single nucleotide differences in alleles. Restriction Mapping - method used to map an unknown segment of DNA by breaking into Solution explanation Single Digests: HpaI: One band at 26 kb indicates a single HpaI site. HindIII: Four bands at 13 kb, 6 kb, 4 kb, and 3 kb indicate multiple HindIII sites. Combination Digest: HpaI and HindIII: Four bands at 7 kb, 6 kb, 4 kb, and 3 kb. Analysis: The HpaI site must be within the largest HindIII fragment (13 kb) because the combination digest does not have a 13 kb band. We know the 6 kb, 4 kb, and 3 kb bands are unchanged in the combination digest, so they are separate fragments with only HindIII sites. The combination of 7 kb and 6 kb equals 13 kb, which suggests that the 13 kb HindIII fragment is split by the HpaI site. This would cut the plasmid at both sites and yield three fragments. The sizes of these fragments would be 0.2 kb, 0.26 kb (0.46 kb 0.2 kb), and 25.54 kb. → Double Digestion with EcoRI and PstI: This would yield three fragments: 0.46 kb, 1.48 kb (1.94 kb - 0.46 kb), and 24.06 kb. Double Digestion with BamHI and PstI: This would yield three fragments: 0.2 kb, 1.74 kb (1.94 kb - 0.2 kb), and 24.06 kb. Steps to create plasmid map:1) Digestion with EcoRI produces a single fragment of 20 Kb. This means that the plasmid is 20 Kb in size and contains only one restriction site for EcoRI. → Digestion with EcoRI: The EcoRI site divides the plasmid into two fragments: one of 0.46 kb and the other of 25.54 kb (since the total size is 26 kb and there's only one EcoRI site). → Digestion with BamHI: The BamHI site would similarly divide the plasmid into two fragments: one of 0.2 kb and the other of 25.8 kb. → Digestion with PstI: The PstI site would yield two fragments: one of 1.94 kb and the other of 24.06 kb. Double Digestion with EcoRI and BamHI: 2) Digestion with BamHI produces 3 fragments. Thus, the plasmid contains three restriction sites for this enzyme. (The first cleavage by the enzyme will linearize the plasmid DNA. The second cleavage will produce two fragments and the third cleavage event will clear one of the fragments in two thus, yielding three fragments in total). According to the fragment sizes, restriction sites will be placed on the plasmid. 3) Digestion with both the restriction endonucleases should produce fragments whose total size should add up to 20 Kb i.e. the size of the plasmid. In this case, the size of three fragments adds up to 17 Kb. This is because there will be two fragments of 3 Kb. 6. The agarose solution is left undisturbed. The gel consists of hydrogen bonded agarose molecules forming a porous matrix to which the DNA molecules move. 7. DNA samples are mixed with a loading buffer and loaded onto the gel. The loading buffer consists of glycerol, which increases the density of DNA samples and helps them settle at the bottom of the well. They also contain dyes, which monitor migrating DNA bands. Agarose Gel Electrophoresis 8. Negatively charged DNA migrates to the positive electrode. Larger DNA molecules move more slowly than smaller molecules allowing size separation. Size depends on agarose concentration. Higher agarose concentration = smaller pore size. Agarose Gel Electrophoresis is a technique commonly used to separate DNA molecules by their size. → Integration: DNA Isolation and Restriction Enzyme Analysis 1. Agarose, a polysaccharide derived from red algae, is added to a buffer such as tris-acetate EDTA (TAE) or Tris-borate EDTA (TBE) and dissolved by heating. → DNA extraction is the removal and the purification of DNA from dells. First, cells are lysed: broken open by a combination of physical and chemical treatment, the treatment of detergents which dissolves cell and nuclear membrane. 2. 1 gram/100 mL = 1% agarose solution. 3. To stain the gel, a fluorescent dye such as ethidium bromide is often added to the agarose solution. 4. The solution is poured into horizontal modes. 5. A comb is inserted into the gel cast to create wells to which DNA samples can be loaded. → SDS or sodium dodecyl sulfate is a commonly used detergent that works by solubilizing proteins and lipids that make up these membranes. → DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. → Salt is added which stabilizes the negative charged phosphate groups in the backbone of the DNA, and after the addition of ice-cold alcohol, precipitates the DNA out of the solution. → White precipitate is collected by spinning in a centrifuge which settles in the bottom of the tube. → Washing and resuspension. → DNA Fingerprinting – can identify novel patterns in DNA. From smaller techniques of molecular analysis, you can combine techniques to form some DNA sequencing techniques. 2. To remove proteins, enzymes such as proteinase K or peptidase K or lysozyme are added to supernatant - Breaks peptide bonds 3. DNA is recovered from the supernatant through precipitation (addition of alcohol and salt; sodium acetate) - Integration DNA Isolation and Restriction Enzyme Analysis II. DNA Extraction and Polymerase Chain Reaction DNA Isolation DNA is isolated in cells is cut in precise positions for many applications (e.g. recombinant DNA technology) ○ Different types of DNA Extraction are used for different cell types 1. Cell lysis 2. Protein removal 3. DNA recovery 1. Inside eukaryotic cells, DNA is packed within the nucleus. Both cell membrane and nuclear membrane need to be ruptured to isolate DNA mechanically by breaking down cells through grinding/sonication or chemically by using detergents and enzymes to dissolve parts of the CM. - Once released, debris are separated from soluble components using centrifugation. - Supernatant: contains nucleic acids and water-soluble proteins. Isolated precipitate is dissolved in water or a buffer. Removal and purification of DNA from cells. 1. Cells are lysed - broken usually though combination of physical disruptions mixed with chemicals (e.g. detergent) - Dissolve cell in the nuclear membrane - SDS (sodium dodecyl sulfate) - Commonly used - Stabilizes proteins and lipids making up membrane = content more freely in the surrounding solution. 2. DNA must be separated from other molecules present - Proteinase K - break down peptide bonds and digest contaminated proteins; added to solution. - Salt - added to sample, stabilizes negatively charged backbone - Alcohol - after addition of ice cold alcohol, precipitates DNA out of the solution -> DNA Precipitation - White Precipitate - collected and spun in a centrifuge where it settles at the bottom of the tube. DNA Fingerprinting Can identify novel patterns of DNA specific to an individual - - Involve R.E. interact with DNA Recognize specific DNA sequences Once specific sites are identified, they cut the DNA resulting in the strand being cut in 1 or more linear places. If DNA extracted was a Plasmid ○ Circular piece of DNA, often found in bacteria ○ Any cuts will result in linear DNA fragment/s. Different R.E. recognize different DNA sequences - Using a combination of these = distinct fragments. In DNA Fragmenting, we can examine fragments using DNA gel electrophoresis. Gel preparation 1. Powdered agarose with buffer 2. Heated until dissolved 3. Nucleotide stain is added to warm mixture 4. Solution is inserted to form the wells 5. Wall com is inserted to form walls 6. Once solid, gel is transferred to gel box filled with buffer and comb is removed 7. Sequence mixture of dyed DNA fragments of no length; DNA ladder is added to each and dyed DNA samples are loaded to the remaining wells. 8. Connected to power source DNA is not typically visible during electrophoresis. ○ Migration of (-) to the (+) side; cathode, anode ○ Smaller pieces move more quickly than larger fragments 9. Once complete, gel is exposed to UV to visualize nucleotide strain in DNA samples Presence can be confirmed based on relative location of lateral bands. Polymerase Chain Reaction Primers - short stretches of nucleotide sequence to initiate DNA synthesis → help identify unique part of genome → single stranded → Forward and reverse primers: the direction is always 5’ to 3’. Forward Primers: will attach to the antisense. Reverse Primers: will attach to the sense strand at the 3’ portion. Problem A DNA with the sequence : 5’CGCATCGATCATGCCCTGA….AGCCCA TTAGATGCC3’. Needs to be amplified using PCR What is the sequence the best reverse primer will have? 5’GGCATCTA3’ 1. Heat mixture to denature protein 2. Reaction is cooled and primers will anneal to homologous regions 3. Once bound, the reaction is heated at the optimal temperature to bind the polymerase. 4. The polymerase recognizes the primer DNA complex and begins synthesis of the new strands using DNTPs in the solution 5. Reaction is heated and proceeds as described across additional cycle → following the 3rd cycle There are eight copies of the gene 6. After the 4th cycle: 16 copies; 5th cycle: 32 copies etc. → After 30 cycles, there are 1,073,741,824 III. DNA Sequencing and Phylogenetic Analysis Sanger Sequencing - Retaining entire nucleotide sequence of an organism. Also called di-deoxynucleotide sequencing. - Pure template sequence is PCR amplified in the presence of appropriate primers and dNTPs, as well as modified bases called di-deoxynucleotides (ddNTPs) → lack a hydroxyl group of deoxynucleotides which limit their ability to form phosphodiester bonds with adjacent nucleotides. → each deoxynucleotide is also labeled with a different fluorescent label to make its detection easier. sequencing from different species or group of individuals → Also known as High throughput sequencing → High proprietary technology → Sequences DNA and RNA in a rapid and cost-effective manner. → can be used for population genetics and clinical studies → fast and cost-efficient Most popular: Reversible Terminator sequencing Others: Pyrosequencing, sequencing by ligation Steps → Fragmentation - Genomic DNA is cut into smaller fragments of 100-1000 base pairs → Ligation of DNA Adapters adapters called Special oligonucleotide sequences with primer sets then ligated to both ends of DNA fragments 1. Denature template DNA into a single-stranded DNA 2. AS primers bind to region of inherit, DNA polymerase starts at a new nucleotide to the DNA strand and occasionally operates a ddNTP instead of a dNTP - Terminates DNA amplification - DNA fragment of varying lengths = each termination with a ddNTPs DNA fragments are run on a capillary gel to separate them on the basis of size Emission spectra of each of these fragments are analyzed through software Next-Generation Sequencing - collective name for group of technologies that can facilitate the relatively rapid generation of → Binding to Chip (Flow Cell) - The single-stranded DNA fragments are loaded to specialized chips called a flow cell with short ○ Precoated oligonucleotide sequences DNA fragments bind to the chip through their adapters. → Denaturation and DNA Amplification - in the amplification stage, DNA polymerase is used to synthesize DNA of a complementary strand. → Washing - The double stranded DNA is denatured and the original template washes off → Bridge Building Remaining complementary strand bends over and forms a bridge-like strand using the second adapter to the other end.