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Bioengineering & Molecular Technology

⁃ Intro:
⁃ genetic engineering: manipulation of DNA in organisms
⁃ recombinant DNA technology: techniques used to engineer genes
⁃ these manipulations have 2 main purposes
⁃ to amplify DNA sequences for further analysis
DNA profiling
ancient DNA
research & discovery of gene function
for use in genetic engineering
⁃ to alter gene expression in an organism of interest for medical,
agricultural, scientific or industrial purpose
⁃ The Polymerase Chain Reaction (PCR): amplification of DNA is primarily accomplished w/
PCR
⁃ PCR is an in vitro (isolated from an organism- in labs) DNA synthesis reaction in
which a specific DNA sequence is replicated over & over again
⁃ PCR uses the natural process involved in DNA replication, & its machinery
(specifically DNA polymerase) to mimic replication of DNA under controlled lab
conditions
⁃ this technique generates many identical copies of a particular DNA sequence
⁃ Requirements of PCR:
⁃ PCR is possible only when DNA sequence info surrounding the gene of interest is
available (b/c PCR requires primer sequences that match the DNA on either side
of the gene)
⁃ one primer is complementary to a sequence on one strand upstream of the
target DNA & the other primer is complementary to a sequence on the other
strand downstream of the target
⁃ the primers will bind to single-stranded target DNA
⁃ primers come from experimenter (artificial)
⁃ Steps of Polymerase Chain Reaction:
⁃ a reaction mix containing dNTPs (deoxynucleotide triphosphate), a DNA
template, copies of the 2 primers, & Taq (DNA polymerase)
⁃ Taq polymerase: an enzyme
⁃ Denaturation: heating the mixture to 95 degrees (C) separates the 2 strands of
the DNA
⁃ Primer annealing: cooling the mixture allows the primers to bond, or anneal to
complementary sections of single-stranded target DNA
⁃ Extension: heating the mixture to 72 degrees (C) causes the Taq polymerase to
synthesize the complementary DNA strand from the dNTPs, starting at the
primer

steps 2-4 are continually repeated on the new DNA to yield the necessary
number of copies
⁃ Gel Electrophoresis (agarose gel): used to separate DNA by size so it can be visualized
⁃ DNA is ran through agarose gel
⁃ DNA has a negative charge
⁃ to move DNA through the gel we attract it to a positive electrode
⁃ shorter pieces will move through the gel easier & quicker
⁃ DNA can also be extracted from the gel
⁃ DNA sequencing: a modified form of PCR
⁃ instead of putting in raw nucleotides a small subset of dideoxyribose is also
added
⁃ dideoxyribose differs from deoxyribose b/c it has no hydroxyl group (just a
hydrogen group)
⁃ dideoxynucleotide stops the extension of the growing strand of the DNA
⁃ PCR reaction in DNA sequencing reactions:
⁃ Primer for replication
⁃ 4 reaction mixtures are prepared: a diff
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⁃ wherever a dideoxynuc
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by
their unique DNA profiles
⁃ one major method of DNA profiling used today is based on PCR & uses short
tandem repeats (STR): repeated sequences of DNA we are aware of
⁃ ex: CGCGCGCG or CAGCAGCAG (repeated sequences of DNA)
⁃ STRs (also called microsatellites) are highly polymorphic regions of DNA that
have short repeated sequences of DNA
⁃ in North America, a group of 13 sites for STR in human DNA are used to generate
a DNA profile for a given indiv
...
will have a given allele for an STR at a given site in their DNA; each
site (called a locus, plural loci) has multiple alleles (diff
...
CGCGCGCGCGCGCG
the distinction is found in the number of repeats (some ppl may have
only 3 repeats in their sequence)
Recombinant DNA Technology:
⁃ recombinant DNA: DNA that results from the use of laboratory methods to bring
together genetic material from multiple sources, creating DNA sequences that
aren’t naturally found in biological organisms
⁃ molecular cloning: is the lab process used to create recombinant DNA
⁃ a set of experimental methods that are used to assemble recombinant
DNA molecules & to direct their replication w/ in host organisms
⁃ PCR & cloning accomplish the same thing, the replication of DNA; PCR does so in
vitro, cloning does so in live organisms
Using Plasmids in Cloning:
⁃ many diff
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palinidromic (same forward & backwards)
⁃ the 1st step in cloning genes into plasmids is to cut the plasmid & the DNA w/
the same restriction endonuclease
⁃ restriction endonuclease’s often make staggered cuts in the DNA,
resulting in sticky ends (complementary single-stranded ends)
⁃ the sticky ends of the plasmids & DNAs will bind by complementary base pairing
⁃ DNA ligase then seals the recombinant pieces of DNA together
Transformation: the process of taking up DNA from the env
...
DNA sequences are cut w/ the same restriction
endonuclease, the presence of the same sticky ends in both samples of DNA
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allows the resulting fragments to be spliced together by complementary base
pairing
⁃ this is the nature of recombinant DNA tech- the ability to create novel
combinations of DNA sequences by cutting specific sequences & pasting them
into new locations
The Potential of Gene Therapy:
⁃ gene therapy: the introduction of a gene into affected cells to replace or
augment defective copies of the gene w/ normal alleles
⁃ in gene therapy, the healthy allele must be sequences & well understood, & then
the DNA has to be introduced in a way that ensures expression of the gene in the
correct tissues, in the correct amount, at the correct time
Introducing Novel Alleles into Human Cells:
⁃ the current vector of choice in gene therapy are retroviruses (these are viruses
w/ an RNA genome)
⁃ they include the enzyme reverse transcriptase (an enzyme that can turn
an RNA molecule back in DNA)
⁃ if human genes are packaged into retrovirus the virus is capable of inserting the
human alleles into a chromosome in a target cell
⁃ in this process first the gene that wants to be replaced will be packed into virus
& the recombinant RNA is prepared
⁃ then the target cell is infected (RNA of virus & enzyme is injected into cell)
⁃ next the RNA is turned into DNA
⁃ reverse transcriptase takes place
⁃ lastly that DNA is incorporated into a humans genome
Using Gene Therapy to Treat X-Linked Immune Deficiency:
⁃ gene therapy has been used to treat severe combined immunodeficiency (SCID)
a fatal genetic disease which causes people to have a weakened immune system
⁃ the type of SCID treated is called SCID-X1 b/c it is caused by mutations in a gene
on the X chromosome
⁃ the gene responsible for SCID-X1 encodes a receptor protein needed for
development of T cells
⁃ a retrovirus engineered w/ a normal receptor gene was used to infect cells from
bone marrow that produce T cells
Ethical Concerns over Gene Therapy:
⁃ it is highly experimental, expensive, & intensely controversial
⁃ it requires many years of additional research & testing, as well as the refinement
of legal & ethical guidelines
Biotechnology in Agriculture:
⁃ most strategies for genetic engineering in agriculture focus on 1 of 3 objectives
⁃ reducing herbivore damage (things that eat plants)
⁃ making crops more resistant to herbicides
⁃ improving the quality of food products
⁃ going into plant & looking @ gene sequence of that gene you’ll find……

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