Chain Termination Sequencing
Biochem - Lecture 10 - Nov. 29th Case Study (con’t) Chain Termination Sequencing: • two methods were developed in the early 1970’w to sequence DNA • chemical sequencing (Maxam and Gilbert) • dideoxy or chain termination sequencing (Sanger) • involves the enzymatic synthesis of a DNA strand in the presence of base specific chain terminations 2’, 3’ Dideoxynucleotides: • Figure 10.20 • dideoxy NTPs act as chain termination • dideoxy lacks 3’ OH so it can’t add another nucleotide • stops the chain - chain terminators • key to chain termination sequencing Materials Required to Sequence a DNA: • DNA to be sequences (template) • oligonucleotide primer that anneals to the template • DNA polymerase • dNTPs (G, A, T, C) • ddNTPs (small amount - 0.2% of dNTP) Extension Reaction in the Presence of the Dideoxy: • Figure 10.20 • 1 in 500 chance dideoxy will be incorporated at that spot, and chain will be terminated • ie - at each base where a dA should be inserted into the growing chain, there is a 1/500 chance that a ddA will be inserted and the chain terminated • a collection of DNA molecules is made which all ahve the same 5’ end, but they differ at the A at which they stop • denature DNA molecules, analyze the products by gel electrophoresis and read the DNA sequence as a ladder from 5’-3’
• autoradiography of a sequencing experiment; typically about 300 bases can be read on sequencing gel • automated DNA sequencing: each ddNTP is labeled with a different dye that fluoresces at a different wavelength • Question: If you added too little of the ddNTPs to a sequencing reaction what would happen? • Answer: the chains would “never” stop and no sequence could be read • Question: To sequence YFG in the plasmid the primer(s) can have a sequence that anneals to the plasmid? • Answer: true • Question: Which of the following need to be considered when expressing your favorite human protein in E. coli? • Answer: cDNA does not have a promoter, different mechanisms of translational initiation, and different protein modification occurs in prokaryotic and eukaryotic cells • cDNA not having a promotor and the different mechanisms are handled through the design of the plasmid used for cloning Plasmid for Expressing YFP in E. coli: • Figure 10.24 • promotor driving your gene; bacterial promotor that you insert before inserting YFG • contains strong promotor • ribosome binding site and ATG downstream from promotor • right after ATG, there is a useful cloning site into which you can insert YFG Production YFP: • purify protein from E. coli • check protein for purity, activity and toxicity • YFP may be subject to proteolysis in E. coli • you believe that an aspartic acid residue with YFP may be leading to the proteolysis • to overcome this you want to change this codon to an alanine Site-directed Mutagenesis: • how do we create a gene with altered sequence and in turn altered function?
Converting a codon for Aspartic acid to a codon for Alanine: • Figure 10.34 • anneal/hybridize oligonucleotides to the single stranded plasmid • transform into E.coli • some will have wild type gene, some will have mutant gene (about 50/50) • can generate the mutation within YFG Transgenic Organisms: • Genetically Modified Organisms (GMO) • definition: an organism that has had its genome permanently altered by genetic engineering • very valuable in research and biotechnology • there are three different types of genetic changes are possible: • gene replacement (only mutant gene is active) • gene knockout (no active gene present) • gene addition (both genes are active) • Figure 10.35 • gene knockout is an easy model for organisms and is a definitive test for the function of a gene/protein (biotech ex. non-spoiling tomato) • gene addition is the basis of the biotech industry and creates an organism that expressed a novel protein (ex. golden rice) • gene replacement allows for examination of the function of an altered gene, or creation of a gene with altered features (biotech ex. nonallergenic peanut) Uses of PCR: 1. Rapid isolation of YFG 2. Analysis of bacteria or viruses in clinical or environmental samples (due to sensitivity of PCR) 3. Diagnosis of genomic disease - or in genetic counseling 4. Forensics - DNA fingerprinting VNTRs (or STRs): • in many regions of our genome there are repetitive sequences (CACACACA...) • the repeat varies from approx. 4-40 times depending on the allele
• the repeat sequences are call Variable Number of Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs) • Question: If a PCR reaction is performed using genomic DNA from one person and a pair of primers flanking one VNTR locus, how many bands will usually be seen after gel electrophoresis? • Answer: 2 - since our genome is diploid there will be a separate PCR products on the gel (paternal gene and maternal gene) • Question: There is a chance that any tow individuals will have the same two bands when any one VNTR locus is examined by PCR? • Answer: True - for this reason, multiple VNTR loci are analyzes in a DNA fingerprint (each VNTR locus requires a distinct pair of primers) Points to Consider with DNA Fingerprinting: • the high sensitivity makes the approach powerful but means extreme care must be taken not to contaminate samples • is is based on probability; that is two individuals could have the same pattern by chance ←
• autoradiography of a sequencing experiment; typically about 300 bases can be read on sequencing gel • automated DNA sequencing: each ddNTP is labeled with a different dye that fluoresces at a different wavelength • Question: If you added too little of the ddNTPs to a sequencing reaction what would happen? • Answer: the chains would “never” stop and no sequence could be read • Question: To sequence YFG in the plasmid the primer(s) can have a sequence that anneals to the plasmid? • Answer: true • Question: Which of the following need to be considered when expressing your favorite human protein in E. coli? • Answer: cDNA does not have a promoter, different mechanisms of translational initiation, and different protein modification occurs in prokaryotic and eukaryotic cells • cDNA not having a promotor and the different mechanisms are handled through the design of the plasmid used for cloning Plasmid for Expressing YFP in E. coli: • Figure 10.24 • promotor driving your gene; bacterial promotor that you insert before inserting YFG • contains strong promotor • ribosome binding site and ATG downstream from promotor • right after ATG, there is a useful cloning site into which you can insert YFG Production YFP: • purify protein from E. coli • check protein for purity, activity and toxicity • YFP may be subject to proteolysis in E. coli • you believe that an aspartic acid residue with YFP may be leading to the proteolysis • to overcome this you want to change this codon to an alanine Site-directed Mutagenesis: • how do we create a gene with altered sequence and in turn altered function?
Converting a codon for Aspartic acid to a codon for Alanine: • Figure 10.34 • anneal/hybridize oligonucleotides to the single stranded plasmid • transform into E.coli • some will have wild type gene, some will have mutant gene (about 50/50) • can generate the mutation within YFG Transgenic Organisms: • Genetically Modified Organisms (GMO) • definition: an organism that has had its genome permanently altered by genetic engineering • very valuable in research and biotechnology • there are three different types of genetic changes are possible: • gene replacement (only mutant gene is active) • gene knockout (no active gene present) • gene addition (both genes are active) • Figure 10.35 • gene knockout is an easy model for organisms and is a definitive test for the function of a gene/protein (biotech ex. non-spoiling tomato) • gene addition is the basis of the biotech industry and creates an organism that expressed a novel protein (ex. golden rice) • gene replacement allows for examination of the function of an altered gene, or creation of a gene with altered features (biotech ex. nonallergenic peanut) Uses of PCR: 1. Rapid isolation of YFG 2. Analysis of bacteria or viruses in clinical or environmental samples (due to sensitivity of PCR) 3. Diagnosis of genomic disease - or in genetic counseling 4. Forensics - DNA fingerprinting VNTRs (or STRs): • in many regions of our genome there are repetitive sequences (CACACACA...) • the repeat varies from approx. 4-40 times depending on the allele
• the repeat sequences are call Variable Number of Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs) • Question: If a PCR reaction is performed using genomic DNA from one person and a pair of primers flanking one VNTR locus, how many bands will usually be seen after gel electrophoresis? • Answer: 2 - since our genome is diploid there will be a separate PCR products on the gel (paternal gene and maternal gene) • Question: There is a chance that any tow individuals will have the same two bands when any one VNTR locus is examined by PCR? • Answer: True - for this reason, multiple VNTR loci are analyzes in a DNA fingerprint (each VNTR locus requires a distinct pair of primers) Points to Consider with DNA Fingerprinting: • the high sensitivity makes the approach powerful but means extreme care must be taken not to contaminate samples • is is based on probability; that is two individuals could have the same pattern by chance ←
