Biochem Basic Molecular Techniques

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Cuts strands of DNA into smaller pieces

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Restriction Enzymes

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Restriction enzymes are also called as

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Restriction endonucleases

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Restriction enzymes recognizes and cuts DNA at specific sequences called?

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Restriction sites

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3 tyoes of cleavage commonly used by restriction enzymes

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- 5' overhang

- Blunt end

- 3' overhang

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The ends of restricted DNA molecules can be joined together by?

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Ligation

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Why does ligation is usedin joining ends of restricted DNA molecules?

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To create new recombinant DNA

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Makes many identical copies of the gene

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DNA Cloning

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To clone a gene, you first need to put gene into a?

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Vector

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Examples of vector

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Plasmid or virus

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Helps carry DNA to the cell

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Vector

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A collection of DNA Fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study

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DNA Library

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3 steps of PCR

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- Denaturation

- Annealing

- Extension

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Denatures the DNA to single strand at 94°C

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Denaturation

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Denaturation occur at what temperature

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94°C

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Rapidly cools the DNA and anneal primers to complementary single strand sequence flanking the target DNA

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Annealing

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Temperature which Annealing occurs

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37-65°C

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Extends primers using a heat resistant DNA

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Extension

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Temperature wherein extension occurs

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70-75°C

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Example of Heat-resistant DNA

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Taq Polymerase

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Taq polymerasewasderived from

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Thermus Aquaticus

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Used to seperate DNA or RNA fragments by size and reactivity

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Nucleic Acid Electrophoresis

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In gel electrophoresis larger fragments migrate?

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Slowly

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In gel electrophoresis smaller fragments migrate

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Quickly

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Key feature in-------- are used in DNA Sequencing

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DNA Replication

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DNA synthesis occurs in what direction

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5'-3'

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DNA synthesis requires a

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Template and a primer

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Is DNA replication semi-conservative

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Yes

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DNA replication is carried out by an enzyme called

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DNA Polymerase

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DNA synthesis requires a 3'-OH to make the next?

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Phosphodiester bond during DNA Synthesis

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Blocks formation of the next phosphodiester bond during DNA synthesis

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DdNTPs

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Used to visualize the results of the sequencing reaction

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Polyacrylamide gel electrophoresis

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– portion of DNA that is cut

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Restricted DNA

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enzymes that remove and destroy proteins in cell

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Proteinase K

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meaning, the sequences of bases are read similar forward and backward.

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Palindromic

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a DNA from two different organisms; fundamental tool in genetic engineering

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Restricted DNA

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an enzyme that performs the process of ligation

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DNA Ligase

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In PCR how many cycles does it require to produce 1 Billion copies

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30 Cycles

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Two types of primer

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Forward Primer

Backward Primer

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 PCR products  1 billion copies of genes overall

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Amplicons