15 DNA Technology and Genomics

Lecture Outline

I. DNA Cloning

A. Research on bacteriophages laid the foundation for recombinant DNA methods

B. Restriction enzymes are A molecular scissors@

1. Restriction enzymes cut DNA in areas of specific base pair sequences that are palindromic

2. Staggered cuts in palindromic regions leave strands with complementary (A sticky@ ) ends

3. Segments of DNA with A sticky@ ends can be joined with DNA ligases

4. Restriction enzymes vary in the number of DNA bases they recognize

a) A restriction enzyme that recognizes a large number of bases has a low probability of cutting in an inappropriate spot and is useful in research on entire chromosomes

C. Recombinant DNA is formed when DNA is spliced into a vector

1. Bacteriophages or plasmids are common vectors

a) Foreign DNA and vector DNA are cut with the same restriction enzyme, making them useful tools

b) Using antibiotics, the transformed colonies can be identified

2. The vector can be introduced into the host cell by transformation

a) Transformation involves making the bacterial cell wall permeable to the plasmid

b) Plasmids often carry genes for resistance to antibiotics

c) Plasmids can carry a DNA segment of smaller than 10 kb

d) Bacteriophage vectors can carry segments of up to 23 kb

3. Another type of vector is a cosmid cloning vector, which is a combination vector with characteristics of both bacteriophages and plasmids

4. Bacterial artificial chromosomes (BACs) accommodate larger fragments of DNA (up to 200 kg of extra DNA)

a) These are particularly of importance in the processes used in the Human Genome Project

5. Engineered viruses can also be used as vectors in mammalian cells

D. DNA can be cloned inside cells

1. A genomic library contains fragments of all of the DNA in the genome

a) A chromosome library contains all of the DNA fragments isolated from the individual chromosomes of the organism of interest

b) It is easier to isolate a gene of interest from a chromosome library than a genomic library

2. Cloning techniques provide the means for replicating and isolating many copies of a specific recombinant DNA molecule

 

 

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a) After the restriction enzyme cuts the DNA to be cloned, the fragments, all with complementary ends, are of different lengths

b) The restriction enzyme cuts both the plasmid and the DNA of the organism with the gene of interest

c) The 2 types of DNA are mixed, which allows pairing of the complementary ends

d) The gene is then cloned in E. coli

e) The cells are incubated on a medium with antibiotics

f) The selection scheme allows only the cells with the recombinant genes to survive, as plasmids often confer resistance to antibiotics

g) Each recombinant bacterium gives rise to a visible colony that is clonal

3. A specific DNA sequence can be detected by a complementary genetic probe

a) A probe is a radioactively labeled segment of RNA or single-stranded DNA that is complementary to the target sequence

b) The probe hybridizes to the base pairs of the sequence of interest

c) The colony with the hybridized sequence is now radioactively labeled and can be detected by x-ray film

4. A cDNA library is complementary to mRNA and does not contain introns

a) Bacteria cannot remove introns, so reverse transcriptase is used to make a DNA copy of the mRNA

b) The complementary DNA (cDNA) can then be inserted into the DNA of a plasmid or virus vector

c) Comparison of cDNA and genomic DNA allows identification of introns and exons

d) cDNA sequences are useful because they lack introns, and bacteria are therefore able to read them and produce a functional protein product

E. The polymerase chain reaction (PCR) is a technique for amplifying DNA in vitro

1. PCR allows amplification of a small amount of targeted DNA in a short time

a) This was developed by Kary Mullis in 1985, for which he received the Nobel Prize in 1993

2. A DNA sequence is first heated to separate the strands

a) The solution is then cooled

b) It is then exposed to DNA polymerase and specific primers to produce 2 identical strands

3. The process is repeated over and over to produce millions of copies of the original DNA strand

4. A DNA polymerase (Taq) from a thermophilic bacterium (Thermus aquaticus) is useful, as it is not adversely affected by the heating process

5. The use of PRC is virtually limitless: from forensics to analyzing fossils

a) One minor drawback is the sensitivity of this process, and samples can easily be contaminated

b) However, samples with even the tiniest amount of DNA can be amplified and analyzed

 

 

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II. DNA Analysis

A. Gel electrophoresis is the most widely used technique to separate macromolecules

1. DNA and RNA are negatively charged and move through a gel at varying speeds due to different molecular weights (lengths)

2. DNA fragments are often denatured, transferred to a membrane, and incubated with a radioactive DNA probe

a) This resultant A blot@ is used for autoradiography or chemical luminescence and further studies

b) This type of blot is called a Southern blot after its inventor, E. M. Southern

(1) The Southern blot can be used to diagnose genetic disorders

c) Blots used for RNA, separated by electrophoresis, are called Northern blots; the Western blot is used for protein or polypeptide molecules (one well known use is to detect antibodies, such as antibodies to HIV: the test for AIDS)

3. Restriction fragment length polymorphisms (RFLPs) can be used to determine how closely related individuals are to each other

a) The size of the DNA fragments produced when cut by restriction enzymes is different in individuals in a population

b) Fragments are separated by gel electrophoresis, transferred by Southern blot, and then probed

c) This technique has been used for paternity cases, in crime-scene analysis, and for discovery of some mutations in genes

B. A great deal of information can be inferred from a DNA nucleotide sequence from the chain termination method of DNA sequencing

1. This method was developed by Sanger and Gilbert in 1974, for which they received the Nobel Prize in 1980

2. Incorporation of dideoxynucleotides allows the investigator to determine the sequence of bases

a) Dideoxynucleotides are modified synthetic nucleotides that prevent elongation of the DNA strand

b) Reaction mixtures are prepared containing DNA polymerase, radioactively labeled primers, 4 deoxynucleotides, and only 1 of each of the 4 dideoxynucleotides (ddATP, ddCTP, ddGTP or ddTTP)

c) Fragments of varying length are therefore formed in each mixture, which may be separated based on length

3. Much of this sequencing is now automated and can be done rapidly using fluorescent dyes instead of radioactive labels

4. Machines can now decode 1.5 million bases in 24 hours, which has lead to the rapid sequencing of entire genomes of a wide variety of organisms

a) The Human Genome Project, began in 1980, resulted in the sequencing of 3 billion base pairs of the human genome in 2001

b) By 2003, genomes of over 100 organisms were sequenced

c) Information is being kept on large computers in databases, many of which are internet accessible

 

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III. Genomics is the study of an organism= s genome to identify all genes, proteins, and RNA produced and to determine the ways that the genes are regulated

a) Genomics has 3 main areas:

(1) Structural

(2) Functional

(3) Comparative

B. Identifying the protein coding genes is important

1. Most human DNA does not code for proteins

2. Short complementary DNA (cDNA) sequences called expressed sequence tags (ESTs) help identify these genes

3. Working through Internet databases, scientists can match a base sequence to these ESTs

C. To determine the function of a gene, it can be silenced

1. Using RNA interference (RNAi) genes of interest can be shut off

2. Function of that gene is determined by the change in the phenotype

D. Gene targeting also reveals function of a gene

1. A gene may be A knocked out@ or inactivated to determine function

2. Knockout mice are models for studying gene function

a) It takes years to produce new strains of knockout mice

b) An inactivated gene is introduced in mouse embryonic cells

c) Cells with the gene are injected into mouse embryos and the mice develop

E. Mutagenesis screening reveals gene function

1. By exposing male mice to chemical mutagens then breeding them, the phenotypes of the offspring may be observed

F. DNA microarrays are used to study gene interaction

1. Copies of cDNA are spotted onto a grid

2. mRNA from 2 cell populations is isolated and used to make cDNA

3. This cDNA is labeled with different fluorescent dyes

4. The 2 populations of cDNA are added to the grid and allowed to hybridize

5. The grid is scanned with laser lights to determine where hybridization took place

6. The activity of the two cell populations can then be compared

7. This technique can be used to identify disease-causing genes

G. Genome sequences of many species are being studied because of the Human Genome Project

1. Studying the genomes of other species helps us determine the function of essential genes

2. Other species= DNA gives us clues about the evolutionary past and holds clues to the cures for diseases

H. The Human Genome Project will change health care in the future

1. Bioinformatics is the use of computers to help manage, compare, and analyze the large number of base sequences of different species

2. Pharmacogenetics will use the genetic make up of an individual to determine treatment strategies

 

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3. Proteomics is the study of all of a cell= s proteins, including which ones are present in different cells and what the structures of each are like

IV. The applications of DNA technology

A. Genetic tests may now determine if a person has a genetic mutation

B. Gene therapy is the use of specific DNA to treat a disorder by actually correcting a genetic problem

Human insulin produced by E. coli was one of the first engineered proteins

        1. Human growth hormone and many other products are now produced by E. coli
        2. Other recombinant products are TPA, TGF-B, human blood clotting factor VIII, and DNase
        3. Recombinant technologies are increasingly being used to produce safer and more effective vaccines
      1. Additional engineering is required for a recombinant eukaryotic gene to be expressed in bacteria
        1. Introduced genes are not necessarily expressed by bacteria
        2. Combining a strongly expressed bacterial promoter sequence with the gene of interest may increase expression
        3. Introduction of a cDNA copy may improve production
      2. DNA typing, or fingerprinting, has many applications
        1. Crime scene evidence analysis, identification of remains, paternity/maternity testing, studying endangered species, and tracking tainted foods are some of these applications
      3. Transgenic organisms have incorporated foreign DNA into their cells
        1. Viruses may be used as vectors to introduce DNA into animal or plant cells
        2. Transgenic animals can produce genetically engineered proteins
            1. Transgenic animals may be produced by injecting the DNA of interest into a fertilized egg cell or an embryonic stem cell (ES cells)
            2. Some transgenic animals produce a useful protein that can be extracted from their milk (e.g., sheep)
            3. Retroviruses may be used as vectors
        3. Gene targeting, in which a researcher A knocks out@ or inactivates a gene to discover the role of the gene
            1. Much research has been done on mice due to their genetic similarity to humans
            2. This technology is used to introduce genes to ES cells
            3. There are about 3000 strains of A knock out@ mice under current study
        4. Mutagenesis screening involves treating male mice with chemical mutagens and breeding them, scanning for unusual phenotypes
            1. This doesn= t knock out the gene, rather it causes a small change, allowing the researcher to see what the result of a tiny change would be
        5. Transgenic plants are increasingly important in agriculture, and are being developed to be resistant to insect pests, draught, heat, cold, herbicides, and salty, or acidic soil
            1. Crops can be developed that are more nutritious and enriched in vitamins A or B, or beta-carotene; although it is hard to increase protein content
            2. Agrobacterium tumefaciens (the crown gall bacterium) is often used in plant biotechnology
              1. The Ti (tumor-inducing) plasmid is used as a vector
              2. The Ti vector primarily infects dicot plants
            3. Genetic A shotguns@ may be used for monocots
              1. Most of our important agricultural plants are monocots (corn, rice, wheat)
            4. Current research involves manipulation of the chloroplast DNA
              1. Questions remain whether it should be labeled, although the majority of scientists believe that transgenic crops are safe

  1. Safety guidelines have been developed for recombinant DNA technology
    1. Concerns about the accidental release of genetically engineered microbes has been groundless to date (see Lecture Enrichment)
    2. Stringent restrictions exist in areas of biotechnology that are not well known, or where potential for hazards are known