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Pcr

Dr. Hanumantappa B. Nayaka
Dr. Hanumantappa B. Nayaka

PCR is a technique used to amplify a specific region of DNA. It involves using primers that are complementary to the outer regions of the DNA sequence being targeted. During PCR, the DNA is denatured and new strands are synthesized using DNA polymerase. This results in exponential amplification of the target DNA sequence. Primers are short oligonucleotides that are selected to target a specific sequence and initiate DNA synthesis. DNA polymerase is the enzyme that copies the DNA sequence. Restriction fragment length polymorphism (RFLP) involves using restriction enzymes to cut DNA into fragments of different lengths that can then be separated by electrophoresis to identify different organisms based on unique DNA fingerprints.

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Dr. Hanumantappa B Nayaka M.Sc., M.Phil., PhD., PDF(Spain)., MIScT (U K) Asst. Professor Department of Life Sciences Kristu Jayanti College Autonomous Bengaluru-77, Karnataka PCR - Polymerase Chain Reaction
PCR - Polymerase Chain Reaction PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence. The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. This results in the synthesis of new DNA strands which are complementary to the parent template strands. These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.
Pcr
Pcr
Pcr
Primer selection Primer is an oligonucleotide sequence will target a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids For example, there is a 村 chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli.
Primer Specificity Universal amplifies ALL bacterial DNA for instance Group Specific amplify all denitrifiers for instance Specific amplify just a given sequence Forward and reverse primers If you know the sequence targeted for amplification, you know the size which the primers should be anealing across If you dont know the sequence What do you get?
DNA Polymerase DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring in Yellowstone National Park This enzyme is heat-tolerant useful both because it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch more specific replication
RFLP Restriction Fragment Length Polymorphism Cutting a DNA sequence using restriction enzymes into pieces specific enzymes cut specific places Starting DNA sequence: 5-TAATTTCCGTTAGTTCAAGCGTTAGGACC 3-ATTAAAGGCAATCAAGTTCGCAATAATGG Enzyme X 5-TTC- 3-AAG- Enzyme X 5-TTC- 3-AAG- 5-TAATTT 3-ATTAAA 5-CCGTTAGTT 3-GGCAATCAA 5-CAAGCGTTAGGACC 3-GTTCGCAATAATGG
RFLP DNA can be processed by RFLP either directly (if you can get enough DNA from an environment) or from PCR product T-RFLP (terminal-RFLP) is in most respects identical except for a marker on the end of the enzyme Works as fingerprinting technique because different organisms with different DNA sequences will have different lengths of DNA between identical units targeted by the restriction enzymes specificity can again be manipulated with PCR primers
Electrophoresis Fragmentation products of differing length are separated often on an agarose gel bed by electrophoresis, or using a capilarry electrophoretic separation
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  • 1. Dr. Hanumantappa B Nayaka M.Sc., M.Phil., PhD., PDF(Spain)., MIScT (U K) Asst. Professor Department of Life Sciences Kristu Jayanti College Autonomous Bengaluru-77, Karnataka PCR - Polymerase Chain Reaction
  • 2. PCR - Polymerase Chain Reaction PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence. The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. This results in the synthesis of new DNA strands which are complementary to the parent template strands. These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.
  • 6. Primer selection Primer is an oligonucleotide sequence will target a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids For example, there is a 村 chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli.
  • 7. Primer Specificity Universal amplifies ALL bacterial DNA for instance Group Specific amplify all denitrifiers for instance Specific amplify just a given sequence Forward and reverse primers If you know the sequence targeted for amplification, you know the size which the primers should be anealing across If you dont know the sequence What do you get?
  • 8. DNA Polymerase DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring in Yellowstone National Park This enzyme is heat-tolerant useful both because it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch more specific replication
  • 9. RFLP Restriction Fragment Length Polymorphism Cutting a DNA sequence using restriction enzymes into pieces specific enzymes cut specific places Starting DNA sequence: 5-TAATTTCCGTTAGTTCAAGCGTTAGGACC 3-ATTAAAGGCAATCAAGTTCGCAATAATGG Enzyme X 5-TTC- 3-AAG- Enzyme X 5-TTC- 3-AAG- 5-TAATTT 3-ATTAAA 5-CCGTTAGTT 3-GGCAATCAA 5-CAAGCGTTAGGACC 3-GTTCGCAATAATGG
  • 10. RFLP DNA can be processed by RFLP either directly (if you can get enough DNA from an environment) or from PCR product T-RFLP (terminal-RFLP) is in most respects identical except for a marker on the end of the enzyme Works as fingerprinting technique because different organisms with different DNA sequences will have different lengths of DNA between identical units targeted by the restriction enzymes specificity can again be manipulated with PCR primers
  • 11. Electrophoresis Fragmentation products of differing length are separated often on an agarose gel bed by electrophoresis, or using a capilarry electrophoretic separation