DNA Technology

Genetic Engineering

* The direct __**manipulation**__ of genes on DNA
* Has led to new biotechnology in which organisms or parts are manipulated to make useful products
* Allow for the making of recombinant DNA: a DNA molecule made from __**two different sources**__

Extraction Process Steps

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1. Disrupt the plasma membrane with a __**detergent**__
2. Destroy proteins and RNA
3. DNA-containing liquid is separated from the remaining contents with a __**centrifuge.**__
4. DNA is precipitated with alcohol

How DNA is cut out

* DNA is cut out with __**restriction enzymes**__ (restriction endonuclease)
* Enzymes cleave or cut out DNA at specific sites along the DNA strand-- restriction sites
* Causes staggered cuts which creates "sticky ends"

How to create recombinant DNA

* The complimentary sticky ends join to a fragment from another DNA that is cut with the __same enzyme.__
* __**DNA ligase**__ helps to bond to new segments together to create a new recombinant DNA molecule

Gel Electrophoresis

* In order to make use of the fragments made by cleaving DNA, the individual __DNA segments must be__ __separated__
* Gel Electrophoresis is the most common separation technique
* Takes advantage of the __negative charge__ of DNA
* Can also separate other biomolecules

Gel Electrophoresis Steps

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1. Restriction enzymes are used to cut DNA
2. The fragments are loaded into the gel
3. Electrical current is applied
4. The DNA fragments will move through the gel towards the positive end of the charge (move from negetive to postive ends)
5. Fragments move based on their size (larger ones are slower)

* The gel can be strained to visualize the fragments

Southern Blotting

* Used to find a particular sequence in a
sample of DNA
* After separation, the fragments are
transferred to a nylon membrane and
bathed in a solution containing probes
* __Short__ DNA fragments are used to find a
DNA fragment of interest
* The probe is a complement and is tagged with
radioactive or fluorescent dyes
* The probe attaches to the DNA sequence of
interest and "tags" it or makes it visible

Polymerase Chain Reaction (PCR)

* Used to amplify (produce) specific regions of DNA
* Can make billions of copies of a particular DNA
segment __without using cells__
* Often used when samples are __very small__
* DNA sample is incubated with a special
DNA Polymerase, artificial DNA primers to
flank the specific regions, and a supply of
nucleotides

Polymerase Chain Reaction (PCR) steps

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**Polymerase Chain Reaction (PCR) steps**


1. Denaturation - heated to separate strands
2. Annealing - cooled so primers may attach
3. DNA synthesis - warmed so polymerase can
copy strands

(video we watched that rapidly duplicated DNA)

Molecular Cloning

* It involves isolating specific sequences or
genes and making new __copies__ of that
sequence
* Allows for the manipulation and study of
specific genes and their protein products
and non-coding regions
* The cloning of recombinant DNA usually
requires a __vector__ to help insert the new
DNA into a bacteria cell where it can be
copied
(Usually a plasmid or a phage)

Plasmid (bacteria) Vectors

* Vectors are vehicles. They transmit an infectious agent from an infected animal to a human or another animal.
* __Plasmid Vectors are used to clone__ __**SMALL PIECES**__ __of DNA__
* Must have two components
* __Origin of replication__ to allow replication inside
of the bacteria
* __Selectable marker__ - usually antibiotic
resistance; allows presence of plasmid to be
easily identified
* The plasmid is cut, new DNA is inserted, and the plasmid will be introduced into a bacteria cell by __**transformation**__ (bacteria takes in foreign DNA and duplicates with it)
* (E. coli is often used for this process)

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Phage (virus) Vectors

* Phage Vectors are larger and can be used to copy LARGER sequences
* Most common is __**phage lambda**__
* Virus will infect host cell and use it to reproduce; new DNA is incorporated into host genome (lysogenic)
* Phage genome is linear instead of
circular
* (put DNA into a virus and let it reproduce through the lysogenic cycle)

DNA libraries

* Using the processes of __molecular cloning__, a
collection of DNAs can be put together in a
DNA library
* Often a genomic library - a representation of
the entire genome
* DNA is fragmented and __each fragment is
inserted into vectors which are placed into host
cells__
* Each cell contains a single fragment in a plasmid or
each phage contains a single fragment
* All cells or all phages together make up the library

cDNA

* Libraries often contain cDNA
* Complementary DNA produced from an edited RNA transcript
* Requires __reverse transcriptase__


* Made so that the DNA sample
contains __no introns__
* More efficient for expressing
eukaryotic DNA in a bacteria
* %%(reverse transcriptase converts RNA into DNA. That new DNA is called cDNA. c = complementary)%%

Reproductive Cloning

* Used to make clones of an entire
organism
* __**Parthenogenesis**__ - an embryo grows
and develops without fertilization
* Involves __replacing the haploid nucleus
of an egg with a diploid nucleus from
a donor cell__ of the same species
* The egg is then placed in a surrogate
mother for development
* The first cloned animal was __**Dolly the sheep**__
in 1996
* Many animals have since been cloned, but
often exhibit abnormalities
* The age of the DNA may affect the __life
expectancy__ of the clone
* Attempts have been made to clone human
embryos as a source of stem cells
Face resistance due to bioethical issues

GMOs

* __Genetically Modified Organisms__
* Produced by introducing recombinant DNA
into an organism
* If the foreign DNA introduced is from a different species, the organism is considered __transgenic__
* Bacteria, plants, and animals have been
genetically modified since the 1970s for
academic, medical, agricultural and
industrial purposes
* (makes food more resistant to disease, animals bigger, etc)

Transformation

* Transformation of bacteria typically uses __plasmids or viruses as vectors__ (plasmid or virus is inserted into bacteria and let sbacteria reproduce with it)
* Requires an __**origin of replication**__ and a __**selectable
marker**__ (reporter genes) to determine which
cells have been transformed
* Usually antibiotic resistance genes or gfp gene (green
fluorescence)
* Almost any gene can be inserted into
bacteria or yeast cells causing the
cells to produce large amounts of
protein product
* Requires expression vectors with extra
sequences needed for the gene to be
expressed
* Example - bacteria are engineered to
produce human insulin and clotting
factor

Genome Mapping

* Finding the location of genes on each
chromosome
* __Genetic maps__ list genes and locations
* __Physical maps__ represent distance in
nucleotides
* Data is entered into international
databases to make information
accessible

Genome Sequencing

* Determines the DNA sequences of a
piece of or the entire genome
* Once sequenced, genomes can be
analyzed and interpreted
* Important for __genetic disease__ study
* Used to generate __DNA microarrays__
* DNA fragments are attached to a glass
slide or silicon chip and are used to
identify active genes and sequences

Manipulating Genes and Expression

* Synthetic DNA and RNA can be made and
manipulated to create and study effects of
mutations
* Reveals cause-and-effect relationships
Could involve:
* __**Knockout experiments**__ - inactivate genes
* __**Antisense RNA**__ - complementary microRNAs
made to block translation
* __**RNA interference (RNAi)**__ - small, interfering
RNAs are made to break down mRNA

DNA Technology

Using these and many other methods,
biotechnology can be used for:

* Diagnosing disease
* Gene therapy
* Medicine/pharmaceuticals
* Forensics
* Environmental issues
* Agriculture