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What are the three natural molecules and processes scientists use to manipulate DNA?

Restriction enzymes, gel electrophoresis, and DNA ligase.


What do restriction enzymes do?

They cut double stranded DNA molecules into smaller, non-infectious fragments.


How do restriction enzymes work?

By breaking the bonds of the DNA backbone in between the 3' hydroxyl group of one nucleotide and the 5' poshphate group of another nucleotide.


What is the restriction site?

The site at which restriction enzymes cleave specif sequences of bases.


How does methylation affect restriction enzymes?

A methyl group can be attached to a host bacterial DNA's restriction site to prevent restriction enzymes from cleaving when not necessary.


What does gel electrophoresis do?

It separates DNA fragments that have been cut with a restriction enzyme.


How does gel electrophoresis work?

DNA fragments will be placed into a semisolid gel, and an electric field will be applied. The DNA will then move across the gel due to its negatively charged phosphate groups, leading to separation between the small fragments.


What information does gel electrophoresis give us?

The number of DNA fragments, the size of those fragments, and the relative abundance of a fragment.


What does DNA ligase do?

It catalyzes the joining of DNA fragments to form recombinant DNA.


How does DNA ligase work?

It creates phosphodiester bonds between DNA fragments.


What is a genomic library?

A collection of DNA fragments that comprise the genome of an organism.


What is a cDNA library?

A smaller DNA library made only of the genes transcribed in a particular tissue. This library is made from complementary DNA.


How are cDNA libraries made?

mRNA is isolated from cells, and cDNA copies are made by complementary base pairing. This reaction is catalyzed by reverse transcriptase.


What is a stem cell?

An unspecialized cell that divides into specialized cells.


What is an expression vector used for?

They introduce a specific gene into a target cell so that the protein can actually be expressed in the cell.


What is a cloning vector used for?

Cloning a DNA fragment by inserting a plasmid cloning vector into a bacterial host and allowing it to multiply.


What are BAC/YAC (bacterial artificial chromosomes/yeast artificial chromosomes)?

Artificial vectors that can carry a greater amount of DNA than some naturally occurring vectors.


What are reporter genes used for?

They help select or identify the host cell that contains the recombinant DNA (ex: ampicillin).


What is DNA-DNA hybridization?

A fragment of DNA that has a radioactive tag attached to it is allowed to adhere to other pieces of DNA in order find a particular corresponding sequence.


What is a transfer filter?

Placing a filter on a culture plate to make a blot of the colonies that are growing on that plate. A blot is an imprint that allows the filter to pick up the colonies in the same pattern that they were found on the plate.


What is a southern blot?

A process that can identify a gene of interest from a DNA molecule that was separated through gel electrophoresis.


Microarray analysis

Thousands of known DNA sequences are attached to a solid surface, and each genes is labeled with a color. The cDNA from a cell or tissue will then be hybridized to the array of probe DNA in order to determine the set of genes expressed in different cells.


What is colony hybridization?

A process by which a labeled DNA probe is used to scan a DNA library.


What is gene mapping?

Using recombination frequencies to determine the physical arrangement of genes.


What is DNA sequencing?

Using a manual or automated machine to determine the order of bases based on overlapping sequences.


What is vitro mutagenesis?

Mutated forms of 
a gene are reinserted into the original species to look for malfunctions in gene expression.


How does PCR work?

It amplifies a particular segment of DNA without the need of using another organism.


What is denaturation of DNA?

Heating of a double-stranded piece of DNA until the hydrogen bonds
 break and the strands separate.


What is RNA interference?

Using small double stranded RNA, called silencing RNA (siRNA), to target mRNA for breakdown. In doing so, the expression of a undesired protein can be prevented.


What is bottom-up sequencing?

First, DNA is amplified and sheared into small fragments. The DNA is then sequenced. A database will search for sequences that overlap in their reads and then pair them up to form contigs (A set of overlapping DNA segments). The DNA sequence of an entire genome can then be determined.


What is restriction fragment length polymorphism (RFLP)?

DNA is cut by specific restriction enzymes into small fragments. The fragments are run on an agarose gel and then labeled with an RFLP probe in a southern blot. This is used to either determine if two individuals have the same genotype, or if an individual has a particular disease.