Genetics Quiz 3

recombinant DNA technology

the joining together of DNA molecules from two different species

the first recombinant DNA molecules were produced in:

1972

functions of recombinant DNA technology in science:

• to isolate and study the control of gene expression and the consequences of mutations within a gene

• to produce vaccines and protein therapies (insulin, interferon, human growth hormone)

• to produce clotting factors for treating hemophilia

• development of gene therapy

restriction enzymes

cuts DNA at specific sequences of bases called recognition sites

cloning vectors

• small circular pieces of DNA used to carry foreign DNA into a host cell

• most common vectors used:

  • Plasmids = small circular DNA found naturally in bacteria

  • Bacteriophage = a virus that infects bacteria and carry DNA

gel electrophoresis

• a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size in an electrophoretic field

• Procedure

  • an agarose and buffer solution is poured into a plastic tray and a comb is placed on one end

  • the agarose polymerizes into a gel and the comb is removed, forming wells

  • colored DNA samples are loaded into the wells and an electric current is applied

  • charged molecules move through the gel

    • smaller molecules travel farther

Restriction enzymes were first discovered by which scientists?

• Werner Arber

• Hamilton O. Smith

• Daniel Nathans

Restriction enzymes are primarily produced by:

bacteria

EcoRI is a restriction enzyme produced by:

Escherichia coli

Hind III is a restriction enzyme produced by:

Haemophilus influenzae

Recognition sites for restriction enzymes are best described as:

palindromic sequences

recognition sequence for EcoR I:

5’- GAATTC - 3’ (cut is made between G and A)

recognition sequence for Hind III:

5’ - AAGCTT - 3’ (cut is made between A and A)

Restriction enzymes can produce which two types of DNA cuts?

sticky ends and blunt ends

A staggered cut in DNA produces:

sticky ends - sing stranded overhangs that can base pair

what sources of DNA are used to clone into a plasmid?

• Genomic DNA

• Polymerase Chain Reaction (PCR) Product

• Complementary DNA (cDNA)

Genomic DNA

• the entire genome is fragmented and used to create a variety of chimeric plasmids

• creates a genomic library

• produces multiple copies of a gene of interest, each with varying lengths depending on the fragment used

Polymerase Chain Reaction (PCR) Product

• rapid technique used for the amplification of DNA by repeatedly replicating a target sequence

• very fast and targeted

• only the gene you want is amplified

• major benefit: all chimeric plasmids will carry the gene of interest

Complementary DNA (cDNA)

• uses enzyme reverse transcriptase to generate DNA fragments

• reverse transcriptase converts mRNA to DNA

• represents only expressed genes

• major benefit: DNA fragment generated will not contain introns

characteristics of cloning vectors

• can be stably maintained in a host organism

• foreign DNA fragments can be inserted into cloning vectors for cloning purposes

• has an Origin of Replication (Ori)

• has a minimum of two selectable markers

• has a unique restriction site within one of the selectable markers

Origin of Replication (Ori)

• allows vector to be replicated in a bacterial host

selectable markers

• genes that help scientists identify bacteria that contain the vector (aka facilitates screening for recombinant organisms)

• Two types:

  • Antibiotic resistance gene (ampicillin or tetracycline)

  • LacZ gene

Why is it important that a restriction site within a selectable marker be unique?

• ensures the vector is cut only once

• the target DNA can be inserted precisely at that location

vector cloning procedure:

1. cut the DNA of interest and vector with the same restriction enzyme (creates compatible ends so they can stick together)

2. mix DNA and vector together with the enzyme ligase

   1. produces:

      1. reannealed vectors without an insert

      2. two or more DNA fragments stuck together

      3. chimeric DNA where a fragment is inserted into cloning vector

3. a bacterial host like E.coli is transformed to make it competent then plated on selective media (containing an antibiotic)

4. chimeric vectors are identified based on which bacteria survive the antibiotic

5. chimeric vectors are expanded by growth in E.coli

Why is transformation followed by plating on selective media?

To select bacteria that have taken up the vector, usually using antibiotic resistance markers.

procedures used to make E.coli competent

• natural transformation

• chemical transformation

• electroporation

transformation

acquisition of DNA from the environment (requires inserting a plasmid into E.coli)

• transformation is necessary because E.coli does not naturally take up DNA

natural transformation

bacteria that can naturally take up DNA

chemical transformation

• cells are treated with CaCl2 (or similar salts)

• this neutralizes the negative charges on DNA and the cell membrane

• a brief heat shock creates pores that allow DNA to enter

electroporation

• a short electric pulse creates temporary pores in the membrane

• DNA enters through these pores

What is the purpose of IPTG in the selection media?

it activates transcription of the LacZ gene which activates production of enzyme beta galactosidase

What color will E. coli colonies be if the lacZ gene is intact?

blue

X-gal

• a colorless pigment

• if beta galactosidase is function, it cleaves X-gal releasing blue dye

If a DNA insert disrupts the lacZ gene, the resulting colonies will be:

• white

• indicates successful insertion of foreign DNA into plasmid

What is the role of ampicillin or tetracycline in the growth media?

• it selects for bacteria containing plasmids

• bacteria without the plasmid insert are killed

when selecting E.coli with chimeric plasmid, what should the growth media contain?

• IPTG (Isopropyl beta-D-1-thiogalactopyranoside)

• X-gal (5-bromo-4-chloro-3-indoyl-beat-D-galacto-pyranoside)

• Antibiotic (ampicillin or tetracycline)

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what method is used to select E.coli cells containing a chimeric (recombinant) plasmid?

Replica Plating

What is the primary purpose of replica plating?

To transfer the same colony pattern to different media

Why is a master plate used in replica plating?

To maintain the original living colonies

What material is commonly used to transfer colonies during replica plating?

Sterilized velvet

traits

displayed characteristics that are controlled by genes on chromosomes