Cytogenetics: DNA Technology
Recent advances in DNA Technology
DNA cloning Polymerase Chain Reaction (PCR) Agarose Gel electrophoresis DNA sequence DNA fingerprinting Gene Therapy
Purpose of DNA technology
Treat diseases Treat genetic disorders Improve human life Improve food crops
Denaturation
96 ̊C
Restriction Enzyme
A DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site.
Sanger sequencing
Also a wide use for the sequencing of individual pieces of DNA, such as DNA cloning and PCR
DNA fingerprinting
Also used to establish paternity
DNA Technology
Involved in the ability to manipulate genes/DNA
agarose gel electrophoresis
Involves running a current through a gel containing the molecules
PCR tube
designed for single use in PCR applications
Extension
72 ̊C
Sanger sequencing
"the chain termination method"
Steps in DNA cloning
1. Cutting and pasting DNA 2. Bacterial transformation and selection 3. Protein production
Components of PCR
1. DNA sample 2. Primers 3. Nucleotides 4. Taq polymerase 5. Mix Buffer 6. PCR tube
Process of PCR
1. Denaturation 2. Annealing 3. Extension
Uses of DNA cloning
1. biopharmaceuticals 2. gene therapy 3. gene analysis
Annealing
55-65 ̊C
polymerase chain reaction (PCR)
A common laboratory technique used to make many copies of a particular region of DNA.
DNA fingerprinting
A laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation.
Sanger sequencing
A method of DNA sequencing based on the selective incorporation of chain-terminating deoxynucleotides by DNA polymerase during in vitro DNA replication.
agarose gel electrophoresis
A technique used to separate DNA fragments (or other macromolecules such as RNA and proteins) based on their size and charge.
Band
A well-defined line of DNA on a gel.
Gene Therapy
An experimental technique that uses genes to treat or prevent diseases.
- Replacing a mutated gene -Inactivating, or "knockingout", amutatedgene (that is functioning improperly) - Introducing a new gene into the body (to help fight a disease)
Approaches to gene therapy:
Taq Polymerase
It is used to automate the repetitive steps in the polymerase chain reaction technique, an extremely important method of amplifying specific DNA sequences.
Restriction Enzyme
Produce cut ends with short single-stranded overhangs
Annealing
Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Biopharmaceuticals
DNA cloning can be used to make human proteins with biomedical applications, such as the insulin.
Method of Sanger Sequencing
DNA sample covered with: - Primer - DNA polymerase - DNA nucleotides - Four dye-labeled - Chain terminating dideoxy nucleotides
DNA fingerprinting
DNA sample taken from a crime scene is compared with a DNA sample from a suspect.
Breaking the DNA of the genome Sequencing the pieces Assembling the sequences
DNA sequencing requires:
Extension
Raise the reaction temperatures so Taq polymerase extends the primers
Agarose
Gels for DNA separation are often made out of a polysaccharide called _______
Denaturation
Heat the reaction strongly to separate, or denature
DNA cloning
In a molecular biology lab, gene and other small pieces of DNA is what's often cloned.
Gene analysis
In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.
Gene Therapy
In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient's body.
Biopharmaceuticals
Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
25-35 times
PCR cycle repeats ______
Transformation
Plasmid & other DNA can be introduced into the bacteria such as harmless E.coliin labs, in a process called ______
antibiotic resistance gene
Plasmids typically contain ___________which allows bacteria to survive in the presence of specific antibiotic.
Extension
Synthesizing new strands of DNA
DNA sequencing
The process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA.
DNA cloning
The process of making multiple, identical copies of a particular piece of DNA.
Denaturation
This provides single-stranded template for the next step
gene Therapy
This technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery.
agarose gel electrophoresis
To check whether the PCR worked; Technique used to visualize the fragments of DNA
DNA cloning
To make a genetically exact copy of an organism.
polymerase chain reaction (PCR)
Used in many areas of biology and medicine including: Molecular biology research Medical diagnostics Branches of ecology
Sanger sequencing
Used to determine the sequences of many relatively small fragments of human DNA.
Sanger sequencing
Was developed by the British biochemist Fred Sanger and his colleagues in 1977.
gel
a slab of jello-like material
Nucleotides
bases A, T, G, and C, which are essentially "building blocks
Gene Therapy
currently being tested only for diseases that have no other cure.
phoresis
migration or movement
Mix Buffer
provides a suitable chemical environment for activity of DNA polymerase
DNA ligase
seals gaps in the DNA backbone
Primers
short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis.
DNA ladder
standard reference that contains DNA fragments of known lengths.
2-4 hrs
takes about _____ to be copied
DNA sample
the sample DNA that contains the target sequence
Goal of DNA cloning
to insert a target gene
electro
utilizes electricity to make molecules migrate
wells
where the DNA samples will be placed; pocket-like indentations of the gel at one end.
