Degenerate primer
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This document provides guidance on designing degenerate primers that can amplify similar genes from multiple species. It recommends selecting a conserved 6-7 amino acid peptide sequence and designing a 20 nucleotide oligo to match. The oligo should accommodate all possible codons for conserved residues among the target proteins. Amino acids with many codons like leucine and arginine should be avoided in favor of those with few codons. Inosine can be used where complete degeneracy exists to reduce the number of primers needed. Degeneracy should also be avoided at the 3' end.
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Primer design
Primers are short DNA sequences used to initiate DNA replication via polymerase chain reaction (PCR). Good primer design is important for PCR to work properly. Primers should be 18-24 base pairs in length and have a GC content and melting temperature that allows for specific annealing. Software tools can help design primers that meet characteristics like avoiding primer-dimer formations and complementarity at the 3' end. Common steps in primer design include specifying the target DNA, selecting primer length and melting temperature parameters, and filtering results.
Sanger sequencing
DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. The Sanger method, developed in 1977, was the most widely used sequencing technique for 25 years. It utilizes chain termination with dideoxynucleotides which lack a 3' OH group, preventing formation of a phosphodiester bond and terminating strand elongation. Four reactions are run in parallel with each dideoxynucleotide labeled with a different color. Gel electrophoresis separates the terminated fragments by size, allowing the DNA sequence to be read by matching fragment sizes to nucleotide colors.
Primer designing
This document discusses primer design for PCR. It begins by defining a primer as a short DNA sequence that is complementary to the target sequence and needed to initiate DNA replication. It notes that primers are essential for PCR, acting like tires on a car. The document then outlines general rules for effective primer design, including having a length of 18-30 nucleotides, a melting temperature of 55-65°C, less than 5°C difference between primer pairs, avoiding primer dimers and secondary structures, having a GC content of 40-60%, and targeting a product size between 150bp to 10kbp.
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Degenerate primers
Degenerate primers are designed from conserved amino acid sequences aligned from multiple species. They contain nucleotide degeneracies that allow binding to related gene sequences. Key steps are:
1) Identifying conserved regions over 5+ amino acids long and within 200-600 bp for primers.
2) Calculating primer degeneracy based on contributing amino acids to minimize values over 64.
3) Optimizing primers by avoiding amino acids with 6-fold degeneracy and adding 5' tails.
Real time PCR
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Polymerase Chain Reaction
This is a powerpoint file of a practical class taken by Dr. Karthikeyan Pethsuamay for the first year MBBS students of AIIMS, New Delhi. Feel free to download and use for educational purposes. Happy learning and teaching!
Don't forget to watch the YouTube video.
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Primer design
Primers are short DNA sequences used to initiate DNA replication via polymerase chain reaction (PCR). Good primer design is important for PCR to work properly. Primers should be 18-24 base pairs in length and have a GC content and melting temperature that allows for specific annealing. Software tools can help design primers that meet characteristics like avoiding primer-dimer formations and complementarity at the 3' end. Common steps in primer design include specifying the target DNA, selecting primer length and melting temperature parameters, and filtering results.
Sanger sequencing
DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. The Sanger method, developed in 1977, was the most widely used sequencing technique for 25 years. It utilizes chain termination with dideoxynucleotides which lack a 3' OH group, preventing formation of a phosphodiester bond and terminating strand elongation. Four reactions are run in parallel with each dideoxynucleotide labeled with a different color. Gel electrophoresis separates the terminated fragments by size, allowing the DNA sequence to be read by matching fragment sizes to nucleotide colors.
Primer designing
This document discusses primer design for PCR. It begins by defining a primer as a short DNA sequence that is complementary to the target sequence and needed to initiate DNA replication. It notes that primers are essential for PCR, acting like tires on a car. The document then outlines general rules for effective primer design, including having a length of 18-30 nucleotides, a melting temperature of 55-65°C, less than 5°C difference between primer pairs, avoiding primer dimers and secondary structures, having a GC content of 40-60%, and targeting a product size between 150bp to 10kbp.
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Bharat Bhushan Negi
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The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
Degenerate primers
Degenerate primers are designed from conserved amino acid sequences aligned from multiple species. They contain nucleotide degeneracies that allow binding to related gene sequences. Key steps are:
1) Identifying conserved regions over 5+ amino acids long and within 200-600 bp for primers.
2) Calculating primer degeneracy based on contributing amino acids to minimize values over 64.
3) Optimizing primers by avoiding amino acids with 6-fold degeneracy and adding 5' tails.
Real time PCR
Real time PCR allows for monitoring of DNA amplification during polymerase chain reaction (PCR), rather than just at the end. There are two main detection methods: using non-specific fluorescent dyes that bind to double stranded DNA, and using sequence-specific fluorescent probes. Common non-specific dyes include SYBR Green I, while TaqMan probes are an example of sequence-specific probes that use fluorescence resonance energy transfer. Real time PCR has applications in disease diagnosis, microbiology research on food and water safety, and quantifying gene expression levels.
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Primer design is a key step in PCR that requires considering various factors to optimize the reaction. These include primer length, melting temperature, GC content, specificity, and potential for secondary structures. Well-designed primers are unique in targeting a single region, have compatible melting temperatures, and do not form hairpins or primer dimers that could inhibit the reaction. Choosing appropriate primers is essential for successful PCR amplification.
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Whole genome shotgun sequencing
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Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
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The bacteriophage lambda can undergo either the lytic or lysogenic cycle. In the lytic cycle, infection leads to production of progeny phage and lysis of the host cell. In the lysogenic cycle, the phage DNA integrates into the host genome where it remains inactive until induced to enter the lytic cycle. Key genes involved include cI which promotes lysogeny, and cro which promotes lysis. The decision depends on the relative levels of these genes - high cro leads to lysis while high cI leads to lysogeny.
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Yeast vectors are useful for expressing eukaryotic proteins due to yeasts' ability to perform post-translational modifications. Common yeast species used include Saccharomyces cerevisiae, Pichia pastoris, and Schizosaccharomyces pombe. Vectors include integrating, episomal, replicating, centromere, and artificial chromosome plasmids. Vectors are introduced into yeast via transformation or electroporation. Expression is controlled by inducible promoters like GAL or CUP1 in S. cerevisiae and AOX1 in P. pastoris.
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Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
Ion torrent sequencing
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Insertion vector
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This document discusses different methods for ligating DNA fragments with vectors, including DNA ligase, homopolymer tailing, linkers, and adaptors. It provides details on how each method works, such as using DNA ligase to join complementary cohesive ends, adding homopolymeric tails to fragments using terminal deoxynucleotidyl transferase, and how linkers and adaptors can be used to generate sticky ends for ligation. The basic difference between linkers and adaptors is that linkers have blunt ends while adaptors have cohesive ends.
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Degenerate primer
- 1. DESIGNING DEGENERATE PRIMERS • Degenerate primers may be used to amplify DNA in situations where only the protein sequence of a gene is known, or where the aim is to isolate similar genes from a variety of species. • A six or seven residue peptide sequence should be selected, corresponding to an oligo of about 20 nucleotides. • If the oligo is designed to amplify several similar protein sequences, then the most conserved regions of the proteins need to be selected. If some of the residues are not completely conserved, then the oligo sequence will need to accommodate all possible codons of all amino acid residues at that site (e.g. if one protein has an E at a particular site, while all the others have a D, then the corresponding oligo sequence will be GAN – see table overleaf). • The peptide sequence should avoid amino acids that have a lot of codons, such as leucine (L), arginine (R), and serine (S). Instead, aim for regions that are rich in amino acids that have only one or two possible codons (i.e. M, W, C, D, E, F, H, K, N, Q, Y – see table overleaf). • Inosine residues can pair with any nucleotide, and so can be used at sites where there is complete degeneracy (i.e. substitute I for N in the table below). This reduces the number of oligos that have to be synthesised. Note that only some manufacturers use inosine, however. • Try and avoid degeneracy at the 3’ end of the oligo (note that it is not necessary to have whole codons), and especially avoid ending in inosine. • The table overleaf may be used to help with primer design: amino acid amino acid symbol nucleotide sequence (with complement (for designing reverse
- 2. degeneracy) primers) methionine M ATG TAC tryptophan W TGG ACC cysteine C TGY ACR aspartic acid D GAY CTR glutamic acid E GAR CTY phenylalanine F TTY AAR histidine H CAY GTR lysine K AAR TTY asparagine N AAY TTR glutamine Q CAR GTY tyrosine Y TAY ATR inosine I ATH TAD alanine A GCN CGN glycine G GGN CCN proline P CCN GGN threonine T ACN TGN valine V GTN CAN leucine L YTN RAN arginine R MGN KCN serine S WSN WSN Key to symbols: R = A + G Y = C + T M = A + C K = G + T S = G + C W = A + T H = A + T + C D = G + A + T B = G + T + C V = G + A + C N = A + T + G + C