DNA Cloning

What are the common forms of DNA technology?

1. DNA sequencing

2. Polymerase chain reaction

3. DNA cloning

4. Gel electrophoresis

What is gene therapy?

This is a technique used to treat genetic disorders that are caused by a nonfunctional gene. It works be delivering the “missing” gene’s DNA to the cells of the body

PCR is a technique that has the ability to make many copies of what?

PCR can make many copies of DNA sequences

How does DNA cloning work?

DNA cloning-make many copies of the DNA fragment of interest (gene)- involves inserting a target gene into a circular DNA moilecules called a plasmid. The plasmis can be replicated in bacteria, making many copies of the gene of interest. In some cases, the gene is also expressed in bacteria, making a protein

What is PCR?

This is a technique used to amplify, or make copies of, a specific target region of DNA

What does PCR rely on?

PCR relies on a thermostable DNA polymerase (Taq polymerase), and requires DNA primers designed specifically for the DNA region of interest

What allows many copies of the target region to be produced in PCR?

The reactionn is repeatedly cycled through a series of temperature changes, allowing many copies of the traget region to be produced

PCR requires a DNA polymerase enzyme to make new strands of DNA, using existing strands as templates. What is enzyme?

Taq polymerase, after the heat-tolerant bacterium from which it was isolated from

What are the three basic steps of PCR?

1. Denaturation (96 C): Heat the reaction strongly to seperate, or denature, the DNA strands. This provides single-stranded templ;ate for the next step.

2. Annealing (55-65 C): Col the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA

3. Extension (72 C): Raise the reaction temp so Taq polymerase extends the primers, synthesizing new strands of DNA

the resukts of a PCR reaction are visulized (made visable) through what technique?

Gel electrophoresis

What is gel electrophoresis?

This is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it seperates DNA fragments according to size

What is DNA sequencing?

DNA sequencing involvces determining the sequence of nucleotide bases in a DNA molecule

How is DNA cloning done?

It creates identical copies of a specific gene uisng restriction enzymes to cut the desired gene, pasting it into a plasmid, and inserting it into bacteria like E.coli. the bacteria replicate the gene and can produce proteins, like insulin. Antibiotic resistance genes in plasmids help select bacteria containg the desired gene

What cuts out the gene in DNA cloning?

Restriction enzymes

What are the 4 essential features of cloning vectors?

1. Origin of replication

2. Selectable marker

3. Multiple Cloning Sites

4. Reporter genes (lacZ)

What does origin of replication do?

Allows independent replication in host cells

What does a selectable marker (amp) do in cloning vectors?

Allows selection of cells with plasmid (antibiotic resistance)

What does a Multiple cloning site do gor cloing vectors?

This is a region with multiple restriction sites to insert DNA

Reporter genes such as lacZ have what function for cloning vectors?

Reporter genes are used for screening clone (blue-white screening)

What are cloning vectors?

DNA molecules used to deliver genetic material into host cells

What are the types of cloning vectors?

• Plasmids

• Phages

• Cosmids, BACs, and YACs

What is Recombinant DNA technology?

rDNA is a hybrid DNA molecule made by combing DNA from different sources

What are the key enzymes/tools used for rDNA?

• Restrictive endonucleases

• DNA ligase

• Plasmid Vectors

• Host cells

What is the purpose of restriction endonucleases in rDNA?

Cut DNA at specific sequences

What is the purpose of DNA ligase?

Joins DNA fragments together

What is the purpose of plasmid vectors in rDNA?

Circular DNA used to carry and replicate foreign genes

What do host cells (usally E. Coli) do for rDNA?

Provide the machinery for replication