Chapter 7: Studying Genes

What are the five general steps of DNA cloning?

1) Cut the DNA using restriction endonucleases 2) Select a cloning vector capable of self-replication 3) Join two DNA fragments covalently using DNA ligase 4) Move recombinant DNA to a host organism 5) Select or identify host cells that contain recombinant DNA (pg. 217)

What are important characteristics of good cloning vectors?

1) Must have a replication origin 2) Genes for antibiotic resistance 2) Several unique restriction enzyme cut sites 4) Small size to accommodate DNA inserts

Explain how pBR322 is used to clone foreign DNA

1) pBR322 is cleaved at the Amp element by Pstl 2) DNA fragments to be cloned .... (pg. 222)

What is a plasmid? What are they used for?

A circular DNA molecule that replicates separately from the host chromosome. They will often confer resistance to antibiotics or perform new functions for the cell. Plasmids are commonly used as cloning vectors. (pg. 220)

What is a DNA library?

A collection of DNA clones, gathered together for purposes of genome sequencing, gene discovery, or determination of gene function (pg. 224)

What is a screenable marker?

A gene encoding a protein that causes the cell to produce a colored or fluorescent molecule. This allows cells that carry the plasmid to be readily identified (pg. 221)

What is Western Blotting

A method used to identify proteins of interest. It makes use of antibodies to detect the presence of specific proteins.

What is polymerase chain reaction (PCR)?

A process by which genes or other DNA segments can be amplified. It relies on the enzymes called DNA polymerase. We first heat the DNA to separate the two strands. We then add synthetic oligonucleotide primers and cool. We then add DNA polymerase to catalyze 5' to 3' DNA synthesis. We can repeat this process as many times we like (pg. 227)

Electroporation

A process that transiently renders the bacterial membrane permeable to large molecules. It's a method to get bacterial cells to uptake plasmid DNA (pg. 221)

pBR322

A representative plasmid that commonly used for as a cloning vector. It has an ori (origin of replication), genes that confer resistance to certain antibiotics (tetracyclin and ampicillin), several unique recognition sequences for restriction endonucleases, and is small in size (pg. 221)

What is an epitope tag?

A short protein sequence that is bound tightly by a well-characterized, commercially available antibody (the antibody is usually attached to fluorescent molecules).

Green fluorescent protein (GFP)

A target gene fused to GFP gene generates a fusion protein that is highly fluorescent. This allows us to see where proteins go in a cell (pg. 242)

Yeast artificial chromosomes (YAC)

A type of cloning vector. It allows the cloning of very long DNA segments (pg. 223)

Polynucleotide kinase

Adds a phosphate to the 5'-OH end of a polynucleotide, to label it or permit ligation (a kinase is an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule) (pg. 217)

Terminal transferase

Adds homopolymer tails to the 3'-OH ends of a linear duplex (pg. 217)

What is DNA ligase?

An enzyme capable of joining two DNA molecules or fragments

What is a clone?

An identical copy (pg. 216)

Type II restriction endonuclease

Cleaves DNA at specific base sequences

What are expression vectors?

Cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene. A typical example is an E. coli expression vector (pg. 233)

Give an overview of DNA cloning

DNA cloning involves cutting two DNAs with restriction enzymes, ligating the DNA fragments together, and transforming the recombinant DNA into a suitable host cell. pg. 218

What is recombinant DNA?

DNA molecules comprised of covalently linked segments from two or more sources (pg. 217)

Reverse transcriptase

Enzyme that can make DNA from RNA

What are restriction endonucleases?

Enzymes that can cleave DNA into smaller pieces (pg. 217)

DNA polymerase I

Fills single-stranded gaps in duplex DNA by stepwise addition of nucleotides to 3' ends (pg. 217)

What are reporter genes?

In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants so we can detect them. An example is GFP

What are bacterial artificial chromosomes (BACs)?

It's a plasmid vector that's been developed to accommodate the cloning of very long segments of DNA (100,000 to 300,000 bp). They are called chromosomes because they are long (pg 222)

What are selectable markers?

It's what allows us to identify which bacterial cells have taken up plasmids. Selectable markers can either permit the grwoth of a cell (positive selection) or kill the cell (negative selection) (pg. 221)

What are shuttle vectors?

Plasmids that can be propagated in cells of two or more species (pg. 223)

Alkaline phosphatase

Removes terminal phosphates form the 5' end, the 3' end, or both.

What are recognition sequences?

Sequences of DNA that are recognized by restriction endonucleases and then cleaved pg. 217

Difference between Southern, Western, and Northern blotting.

Southern blotting uses gel electrophoresis for the detection of a specific DNA sequence in a sample of DNA. Western blotting also uses gel electrophoresis but it is to detect proteins and separate them based on size and shape. Northern blotting is for the detection of RNA sequences, and so is geared towards detecting gene expression (the technique is very similar to Southern blotting only formaldehyde is used to denature the RNA).

What is a genome?

The amount of genetic information needed to specify an organism (pg. 216)

What is transformation?

The process by which plasmids are introduced into bacterial cells. The cells and plasmid DNA are incubated together at 0° C in a CaCl solution, then subjected to a heat shock.

Complementary DNAs (cDNAs) and cDNA library

These are formed when we isolate mRNA and use reverse transcriptase to synthesize DNA. The resulting double stranded DNA fragments are inserted into a suitable vector and cloned, creating a population of clones called a cDNA library. These are different from DNA libraries because they contain only DNA that is expressed. (pg. 225) (see http://www.majordifferences.com/2013/11/difference-between-genomic-and-cdna.html#.V_FPduArLic)

Genomic library

This is produced when the complete genome of an organism is cleaved into thousands of fragments and ALL the fragments are cloned by insertion into a cloning vector. (pg. 224)

How to make blunt ended DNA ends "sticky"

Treat the DNA with dTTP termianl transferase and dATP terminal transferase

Sanger method

a method of DNA sequencing, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication https://www.youtube.com/watch?v=FvHRio1yyhQ (pg. 228)

What is a cloning vector?

a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Do restriction enzymes make blunt or sticky ends?

find out pg. 217

What are DNA polylinkers?

short stretches of DNA engineered into a vector that provide recognition sites for several restriction enzymes to facilitate DNA cloning.