Download notes File - selu moodle

Chapter 17
Vocabulary:
Recombinant DNA
Restriction endonucleases
Gel electrophoresis
Clone
Vector
DNA library
cDNA
DNA fingerprinting
PCR
Southern blot
Northern blot
Western blot
Gene therapy
Oultine
17.1 Recombinant DNA
Recombinant DNA (DNA that contains information from 2 different sources)
Uses restriction enzymes (restriction endonucleases) to cut DNA at specific sites
Allows for creation of physical maps
Allows for creation of recombinants
Restriction enzymes usually cut at palindromes leaving “sticky ends”
Can also make blunt cuts
DNA ligase can seal recombinants
Bacteria pick up recombinant DNA when the membrane is compromised by
temperature of electrical impulses
Gel electrophoresis uses an electric current to separate DNA based on size.
17.2 Introducing Foreign DNA into Cells
Clone
Vectors carry foreign DNA for cloning
Plasmids
Must have: origin of replication (so bacteria will copy the plasmid)
Selectable marker (to check for successful recombination)
Restriction sites (places DNA can be introduced)
Artificial chromosomes (for larger DNA)
Vectors for eukaryotic cells (no plasmids so harder to accomplish)
Inject DNA into fertilized egg cell
Inject stem cells into an embryo
Gene gun
Bacterial pathogens (tumor inducing plasmids) carrying gene of interest
17.3 Amplifying DNA Without a Vector - PCR
PCR makes millions of copies of a sample of DNA
Requires: DNA of interest (to be sequenced)
Primers (to initiate replication and to bind to DNA of interest)
Deoxyribo nucleotides (to add to growing strands of DNA)
Taq polymerase (to catalyze reaction)
Heating cycles cause dsDNA to separate
Cooling cycles allows primer to bind and DNA polymerase to synthesize DNA
This continues in an automated way for many cycles
17.4 Storing and Sorting DNA Fragments
DNA libraries contain entire genome of an organism contained in vectors
cDNA libraries contain the coding genome of an organism (can be different at different
developmental stages or in different tissues)
mature mRNA transcript
reverse transcriptase
mRNA / DNA hybrid
RNase
Single stranded DNA
DNA polymerase
Double stranded DNA
17.5 Analyzing and Creating DNA differences
Southern blot procedure
Cut DNA with restriction enzymes
Separate DNA fragments with gel electrophoresis
Denature dsDNA
Blot gel onto filter paper
Incubate filter paper with probe (complementary to specific gene)
Expose bands that bound to probe
Uses:
To identify a single gene among thousands of fragments of DNA
Detect sequences of DNA in an organism’s genome
Used in gene discovery and gene mapping
To analyze the genetic patterns in an organism’s DNA
To identify gene mutations, deletions, duplications, and gene rearrangements involved in
diseases
To determine the number of copies of a particular DNA sequence presented in the
genome of an organism
To identify related DNA sequences in the genome and to determine if there is a gene
family
To detect certain cancers and genetic diseases
Used in DNA fingerprinting, genetic engineering, & forensic science for tests such as:
Paternity testing
Personal identification
Sex determination
Species exclusion
Northern blot is used for mRNA detection (does not require Denaturation)
Western blot is used for protein detection (uses antibodies to bind to proteins)
DNA fingerprinting uses restriction enzymes and gel electrophoresis to diagnose disease,
establish biological relationships, identify individuals or in criminal cases
DNA fingerprints are like consumer good barcodes; all a little different
Takes advantage of short tandem repeats that vary in the human genome
We can work backward to determine the gene of interest by using “knock out”
experiments
17.6 Medical Applications
Human genes inserted into bacteria for production
Insulin, interferon, human growth hormone, erythropoietin, etc.
Atrial peptides, tissue plasminogen activators
Subunit vaccines
DNA vaccines
Plant based vaccines
Gene therapy replaces a defective allele with a working copy to overcome disorders /
diseases
RNAi vectors
Viral vectors
Hurtles to gene therapy seem to be insertion points and regulation of the new gene
17.6 Agricultural Applications
Plants get genes to alter their phenotype
Disease resistance, insect resistance, herbicide resistance, drought resistance,
changes in nutritional content, biopharming of useful compounds
Concerns include cross pollination with wild type varieties, loss of biodiversity, long
term effects of consumption of genetically modified food, possible allergic reactions
Domesticated animals are also modified
Improvements in desirable traits, pharmaceutical production in milk,
environmental benefit in waste alteration
Most important information from this chapter:
How to make recombinants using restriction enzymes (plasmids, vectors)
Gel electrophoresis.
PCR
How to make cDNA and why one would want to
Southern, Western, Northern blotting techniques
DNA fingerprinting (they should be able to read a gel to determine if a suspect is guilty.
They should also be able to read a gel to determine paternity.)
Gene therapy
Mention applications in medicine and agriculture as examples not as information to be
memorized.