Cytogenetics: DNA Technology

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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.


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