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    Recombinant DNA Technology

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    hunter kim
    This document discusses recombinant DNA technology, which involves extracting DNA from different sources and joining them together. Some key points covered include: - The goals of rDNA technology include isolating and characterizing genes, altering genes, and understanding hereditary diseases. - The process involves isolating, cutting, joining, and amplifying DNA using restriction enzymes, DNA ligase, vectors like plasmids, and bacterial transformation. - Applications include producing medicines like insulin through bacteria, developing pest-resistant crops, and conducting gene therapy in humans.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd

    Recombinant DNA Technology

    Uploaded by

    hunter kim
    29 views4 pages
    This document discusses recombinant DNA technology, which involves extracting DNA from different sources and joining them together. Some key points covered include: - The goals of rDNA technology include isolating and characterizing genes, altering genes, and understanding hereditary diseases. - The process involves isolating, cutting, joining, and amplifying DNA using restriction enzymes, DNA ligase, vectors like plasmids, and bacterial transformation. - Applications include producing medicines like insulin through bacteria, developing pest-resistant crops, and conducting gene therapy in humans.

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    Recombinant DNA technology

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    This document discusses recombinant DNA technology, which involves extracting DNA from different sources and joining them together. Some key points covered include: - The goals of rDNA technology include isolating and characterizing genes, altering genes, and understanding hereditary diseases. - The process involves isolating, cutting, joining, and amplifying DNA using restriction enzymes, DNA ligase, vectors like plasmids, and bacterial transformation. - Applications include producing medicines like insulin through bacteria, developing pest-resistant crops, and conducting gene therapy in humans.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    29 views4 pages

    Recombinant DNA Technology

    Uploaded by

    hunter kim
    This document discusses recombinant DNA technology, which involves extracting DNA from different sources and joining them together. Some key points covered include: - The goals of rDNA technology include isolating and characterizing genes, altering genes, and understanding hereditary diseases. - The process involves isolating, cutting, joining, and amplifying DNA using restriction enzymes, DNA ligase, vectors like plasmids, and bacterial transformation. - Applications include producing medicines like insulin through bacteria, developing pest-resistant crops, and conducting gene therapy in humans.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 4

    Recombinant DNA technology

    Contents
     Introduction
     Discovery
     Goals and objectives
     rDNA technology procedure
     Enzymes
     Vectors
     Techniques
     Applications
     Summary

    Recombinant DNA technology


    DNA molecules that are extracted from different sources and chemically joined together; for
    example, DNA comprising an animal gene may be recombined with DNA from a bacterium

    Discovery of recombinant DNA technology


     Discovery of DNA structure Watson & Crick in 1953
     Isolation of DNA ligase in 1967
     Isolation of REase in 1970
     Paul Berg generated rDNA technology in 1972
     Cohen & Boyer in 1973 produced first plasmid vector capable of being replicated within a
    bacterial host

    Goals of recombinant DNA technology


    • To isolate and characterize a gene
    • To make desired alterations in one or more isolated genes
    • To return altered genes to living cells
    • Artificially synthesize new gene
    • Alternating the genome of an organism
    • Understanding the hereditary diseases and their cure
    • Improving human genome

    Procedure of making rDNA


    • Isolating of DNA
    • Cutting of DNA
    • Joining of DNA
    • Amplifying of DNA

    Isolating of DNA

    Cutting of DNA
    • DNA can be cut into large fragments by mechanical shearing.
    • Restriction enzymes are the scissors of molecular genetics.

    Restriction enzyme
    • A special class of sequence-specific enzyme
    • Found in bacteria
    • Site-specific-cleave DNA molecules only at specific nucleotide sequence
    • REases recognize DNA base sequence that are palindrome
    • REase make staggered cuts with complementary base sequences for easy circulization

    Joining DNA

    Amplifying the recombinant DNA


    • Transforming the recombinant DNA into a bacterial host strain.
    • The cells are treated with CaCl2
    • DNA is added
    • Cells are heat shocked at 42 C
    • DNA goes into cell by a somewhat unknown mechanism.
    • Once in a cell, the recombinant DNA will be replicated.
    • When the cell divides, the replicated recombinant molecules go to both daughter cells which
    themselves will divide later. Thus, the DNA is amplified

    Amplifying the recombinant DNA

    Enzymes used in recombinant DNA technology

    Vectors used in rDNA technology


    • A vector is an area of DNA that can join another DNA part without losing the limit for self-replication
    • Should be capable of replicating in host cell
    • Should have convenient RE sites for inserting DNA of interest
    • Should have a selectable marker to indicate which host cells received recombinant DNA molecule
    • Should be small and easy to isolate

    Vectors used in rDNA technology

    Plasmid vector
    • Plasmids are small, circular DNA molecules that are separate from the rest of
    the chromosome.
    • They replicate independently of the bacterial chromosome.
    • Useful for cloning DNA inserts less that 20 kb (kilobase pairs).
    • Inserts larger than 20 kb are lost easily in the bacterial cell.

    Lamda phage vector


    • Lamda phage vectors are recombinant infections, containing the phage chromosome in addition to
    embedded "outside" DNA.
    • All in all, phage vectors can convey bigger DNA groupings than plasmid vectors.
    •

    Cosmid vector
    • Cosmids are hybrids of phages and plasmids that can carry DNA
    fragments up to 45 kb.
    • They can replicate like plasmids but can be packaged like phage
    lambda

    Expression vectors
    • Expression vectors are vectors that carry host signals that
    facilitate the transcription and translation of an inserted
    gene.
    • They are very useful for expressing eukaryotic genes in
    bacteria.

    Yeast artificial chromosomes (YACS)


    • Yeast artificial chromosomes (YACS) are yeast vectors that have been
    engineered to contain a centromere, telomere, origin of replication, and
    a selectable marker.
    • They can carry up to 1,000 kb of DNA.
    • they are useful for cloning eukaryotic genes that contain introns.

    Bacterial artificial chromosomes (BACS)


    • Bacterial artificial chromosomes (BACS) are bacterial plasmids derived
    from the F plasmid. They are capable of carrying up to 300 kb of DNA.
    Techniques used in rDNA technology
    • Gel electrophoresis
    • Cloning libraries
    • Restriction enzyme mapping
    • PCR
    • Nucleic Acid Hybridization
    • DNA Microarrays

    Gel electrophoresis
    • Gel electrophoresis – DNA fragments of different sizes can be separated by an electrical field
    applied to a “gel”.
    • The negatively charged DNA migrates away from the negative electrode and to the positive
    electrode.
    • The smaller the fragment the faster it migrates.

    Cloning libraries
    • Libraries are collection of DNA clones in a certain vector.
    • The goal is to have each gene represented in the library at least once.
    • Genomic - made from RE DNA fragments of total genomic DNA
    • cDNA (complementary DNA) – made from DNA synthesized from mRNA

    PCR
    • Allows the isolation of a specific segment of DNA from a small DNA (or cell sample) using DNA
    primers at the ends of the segment of interest.

    Restriction enzyme mapping


    • Frequently it is important to have a restriction enzyme site map of a cloned gene for further
    manipulations of the gene.
    • This is accomplished by digestion of the gene singly with several enzymes and then in
    combinations.
    • The fragments are subjected to gel electrophoresis to separate the fragments by size and the
    sites are deduced based on the sizes of the fragments.

    Nucleic Acid Hybridization


    • A Southern allows the detection of a gene of interest by probing DNA fragments that have been
    separated by electrophoresis with a “labeled” probe.
    • Northern Blot (probe RNA on a gel with a DNA probe)
    • Western Blot (probe proteins on a gel with an antibody)

    DNA Microarrays
    • vast majority of the protein- encoding qualities onto a microarray
    chip, utilizing innovation in light of the DNA silicon chip industry.
    • The chip can be utilized to hybridize to cell RNA, and measure the
    statement rates of a substantial number of qualities in a cell.

    Applications of rDNA technology


    • Agriculture: growing crops of your choice (GM food), pesticide resistant crops, fruits with
    attractive colors, all being grown in artificial conditions
    • Pharmacology: artificial insulin production, drug delivery to target sites
    • Medicine: gene therapy, antiviral therapy, vaccination, synthesizing clotting factors
    • Other uses: fluorescent fishes, glowing plants etc.

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