Download AP Biology DNA Technology: The manipulation of organisms or their

AP Biology
DNA Technology:
The manipulation of organisms or their components to make useful products
Recombinant DNA:



Nucleotide sequences from two different sources, often two species, are combined in vitro (in a test
tube) into the same DNA molecule. Can occur artificially through Recombinant DNA Technology.
Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of
genes for practical purposes
To work directly with specific genes, scientists prepare well-defined segments of DNA in identical
copies, a process called DNA cloning animation
o Gene cloning involves using bacteria to make multiple copies of a gene (amplify)
o Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial
cell.
o Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA
o This results in the production of multiple copies of a single gene.
 This gene must be distinguished from other genes that may have been introduced into
the bacteria. Usually multiply genes are inserted.
o DNA cloning can be used to produce large amounts of proteins such as insulin, tissue
plasminogen activator (TPA- breaks up blood clots), vaccines, etc.

Using Restriction Enzymes to Make Recombinant DNA
o Uses restriction enzymes to cut DNA at specific recognition sites. (these enzymes are obtained
through bacteria) animation
 The cut is usually staggered producing sticky ends. (vs. blunt ends)
 Usually cut at palindrome sites.
o A vector is used to transmit the fragment.
 Usually plasmids because they can be introduced into bacteria through transformation.
 The plasmid is also cut with a restriction enzyme and DNA ligase connects the
segments.

A genomic library that is made using bacteria is the collection of recombinant vector clones produced
by cloning DNA fragments from an entire genome
o These clones can be used to study particular genes or groups of genes..
Scientists can also make a DNA library by using mRNA extracted from cells expressing the gene.
o mRNA is used with a reverse transcriptase to create complementary DNA (cDNA).
o cDNA libraries are often used to study specific proteins.


o Specific DNA sequences can be isolated by hybridization using a Nucleic acid probe.
PCR: used when DNA is scanty or impure. Specific and quick
o
A selected sequence of DNA can be amplified (make many copies)
 DNA is denatured opening the strands.
 Primers are used to bracket selected region (5’ and 3’ sides)
 DNA polymerase extends primers, replicating the DNA
 Repeat over and over to make many copies.
DNA Technology allows us to study the sequence, expression, and function of a gene


Restriction fragments can be separated through Gel Electrophoresis in which the fragments are run
through a gel containing an electrical field.
o DNA runs towards the positive pole because it is negatively charged.
o Smaller segments migrate further than large ones.
o Every individual has a unique set of fragment lengths because of polymorphisms (slight
differences in DNA sequences)
 These fragments are called restriction fragment length polymorphisms or RFLP’s.
 In DNA fingerprinting, RFLP’s are compared, in particular short tandem repeats (STRs)
Restriction Fragment Analysis can be used to study different alleles for a trait.
o Each allele will have a unique banding pattern.
o Can be used to identify carriers (heterozygotes)

Forensic Evidence
o RFLP’s create a unique and specific pattern of bands and can be used as a DNA fingerprint.
 Paternity testing
 A comparison of the DNA of a mother, her child, and the purported father can
conclusively settle a question of paternity.
 A child must share all their bands in common with the mom and dad.
 Crime scene investigation
o Southern Blotting can be used to isolate particular genes being examined.
 Combines gel electrophoresis with DNA hybridization
 Uses a probe which is a radioactive single-stranded DNA molecule that is
complementary to the selected DNA.
 Through capillary action, the radioactive probe is pulled up out of the gel and onto a
sheet of paper.
 A film is placed over the paper. The radioactive probe burns an image of the bands into
the film.
Analyzing gene function






Northern Blotting:
o Procedure is the same as southern blotting, but uses mRNA instead of DNA.
o Used to analyze gene expression changes taking place during different times in development.
 If an mRNA is being made, then that gene is being expressed.
Reverse transcriptase polymerase chain reaction (TR-PCR)
o mRNA + reverse transcriptase = cDNA, which is then run through PCR and gel electrophoresis.
o Like northern blotting, determines what genes are being expressed at different times or in
different tissues.
In situ hybridization
o Same purpose, but probes bind to mRNAs in intact organism.
DNA microarray assays: used to analyze gene expression changes taking place in a large number of
genes.
o A large number of DNA fragments representing different genes are fixed to a glass slide making
a grid. Fluorescent cDNA strands made using mRNA bind to their complementary genes.
Brightness of the tag (spot) displays the level of expression taking place.
In vitro mutagenesis
o Introduces a mutation into a cloned gene. The “knocked out” gene is then inserted and can be
analyzed based upon its effects.
RNA interference (RNAi)


o Also prevents gene expression by blocking the mRNA.
Genome-wide association studies
o Used in humans due to ethical concerns. Analyze genetic differences in a population between
all individuals with a trait or disease and all people without. Compare genomes using genetic
markers, dna sequences that vary in a population.
 Single nucleotide polymorphism (SNP): useful genetic marker, single base-pair
variation in the human genome.
Gene Structure
DNA sequencing: Dideoxy Chain Termination Method
o ddNTP (dideoxyribonucleotide) is a modified nucleotide which terminates a growing strand
because it lacks the attachment site for the next nucleotide.
o ddNTP are fluorescently tagged.
o DNA is denatured, template strand is copied using primer, polymerase, nucleotides, and
ddNTPs which randomly end the growing strand.
o New strands are run through a gel (separating by size) and the fluorescent tags are used to
determine the nucleotide sequence of the complementary strand.
Research and other Applications



Disease Diagnosis
o PCR can be used to amplify genes and thus detect elusive diseases such as HIV, sickle-cell,
hemophilia, cystic fibrosis, Huntington’s and Duchenne’s muscular dystrophy.
Human Gene Therapy: the alteration of an afflicted individual’s genes.
o Can be used to treat disorders traceable to a single defective gene.
o Normal gene is inserted using a viral vector.
 Needs to be a cell that will divide throughout the patient’s life such as a bone marrow
cell.
 Has not been completely effective. (Germ-line cells? Ethics?)
Agricultural
o Animal Husbandry and “Pharm” Animals.
 Transgenic animal: an animal that contains genes from another source.
 Performed by inserting a cloned gene into an egg cell. The gene can even come
from a different species. As the organism develops, all cells will express the gene
and make the desired protein.
 Can be used to “create” animals that are leaner, or more muscular, or have
better wool. Can also be used as a pharmaceutical factory, having animals
produce proteins we need.
o Genetically engineered plants.
 Can insert gene using Ti Plasmid as a vector. Plasmid enters bacteria, then the bacteria
enters the single-celled plant. Plant grows containing the gene. Can make plant
resistant to microbes and herbicides. Can also create “pharm” plants.

Should genetically modified (GM) organisms be created?