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Bio 181: Recombinant DNA Techniques


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Define recombinant DNA.
DNA molecule produced artifically and containing sequences from unrelated organisms.
Define genetic engineering.
Use of techniques involving recombinant DNA technology to produce molecules and/or organisms with new properties.
Define biotechnology.
All inclusive term for several technologies including but not limited to recombinant DNA. Refers to the use of technology in applications for solving fundamental problems in biology.
Define genomics.
Study of genes expressed in cells.
Define proteonomics.
Study of proteins produced in cells.
What are restriction endonucleases? and what do they do?
- They cut double stranded DNA at specific sequences.

- protect against viruses in bacteria.
Sequences in restriction endnocucleases may be palindromes. What is a palindrome?
- a sequence which is the same when read in either direction.
Ex: "MOM"
Explain how gel electrophoresis separates nucleic acids or proteins.
-DNA fragments have a "-" charge

-a porous gel acts as a sieve, holding up big fragments

-fragments take up a dye that fluoresces under UV light
Describe the Southern technique.
-labeled probe complementary to desired DNA sequence

-hybridization can show migration position of known DNA sequence
How is recombinant DNA made?
-EcoR1 cuts on two different strands of DNA

-at lower temperature, the fragments can pair

-DNA ligase (joins Okazaki fragments) can generate recombinant DNA
Describe the steps involved in cloning DNA, including what happens at each of the steps leading to a clone of bacteria with a copy of the desired gene.
-host taking up recombinant DNA can be screened for the presence of recombinant plasmids

-in the example, bacteria that are resistant to ampicillin and sensitive to tetracycline contain recombinant plasmids

-another system uses the LAC operon plus ampicillin resistance in plasmids; recombinants are resistant to amp but fail to make β-galactosidase

-the last step, identifying a clone of bacteria with the desired gene
1. clone- colony grown from a single bacterium
2. DNA probes can screen for clone with gene of interest

Another Summary:
-Vector (plasmid DNA with drug resistance genes) plus DNA of interest cleaved with a restriction endonuclease.
-DNA samples are mixed, allowed to hybridize, and joined by DNA ligase.
-Host cells are made to take up DNA hybrids, and transforments selected by drug resistance.
-Insertional mutagenesis or disruption of a plasmid gene by the insert is used to select for bacteria harboring recombinant DNAs.
-Bacteria carrying recombinant plasmids must be probed or screened to identify the presence of the gene of interest.
How are recombinant DNA introduced into cells?
Plasmid vectors
What are plasmid vectors?
small extrachromosomal DNA (2000-6000 bp) in a bacterial cell which can replicate independently but which cannot integrate into the host chromosome
What are reporter genes?
Drug resistance plasmids that are not essential for the cell's growth, but confer antibiotic resistance.
What does YAC stand for?
yeast artificial chromosome
What plasmids can be used to introduce genes into plants?
Ti plasmids
Define DNA library. How are libraries used?
-cloning fragments of DNA generated from the complete genome can produce a mixture of bacteria which have all of our genes

-cultures of these recombinant DNA containing bacteria constitute a DNA library
How are transcribed genes cloned?
-reverse transciptase, makes a cDNA copy of mRNA

-cloning fragments gives a library of transcribed genes

-since mRNA's were used, library contain only exons
Explain how DNA can be sequenced, including the role of the primer, didoxynucleotides, and gel electrophoresis.
1. A primer sequence is determined for an unknown fragment of single-stranded DNA.

2. This unknown DNA is combined with DNA polymerase, primer, dNTPs and ddNTPs which have been marked with fluorescent dye. If a dNTP is incorporated into the growing strand, replication continues. If a ddNTP is used, however, replication is terminated.

3. Replication is allowed to continue until many different fragments of varying lengths are produced. These fragments are separated by gel electrophoresis. The terminating ddNTP is identified by its color, and the sequence can be "read" from the bottom of the gel to the top.

4. The sequence of the newly synthesized DNA (which is deduced from the gel) is the complement of the unknown strand.
What is the difference between 2-deoxyribose(dNTP) and 2,3-dideoxyribose (ddNTP)?
During sequencing:
If a dNTP is incorporated into the growing strand, replication continues. If a ddNTP is used, however, replication is terminated.
Describe the PCR technique.
-PCR is polymerase chain reaction

1. Primers (DNA fragments) are synthesized that are complementary to both strands of a specific sequence of DNA.

2. Taq DNA polymerase (thermal stable) and the four nucleotide triphosphates are added.

3. DNA strands are denatured by heating to 95o and the solution cooled to 55o to allow the primers to hybridize.

4. The polymerase uses each strand as a template and makes a copy by extending the 3' end of the primer.

-If you heat again and then cool, the process repeats.
Repeating this cycle 35 times gives 2 to the 35 power or 10 billion of copies of the gene.
Gene Inactivation (knockout mouse):
What's its significance, and what are the steps for its use?
-allows testing of gene function, and produces mouse models of human genetic disease.

1. targeted gene on a plasmid inactivated by insertion of a marker gene

2. vector inserted into a mouse stem cell

3. recombination puts the marker/inactivated target in chromosome

4. stem cell transplanted into an early mouse embryo

5. extensive selection and further matings can give knock out mouse
Describe how DNA array studies are performed, including how data is interpreted.
1. cellular mRNA converted to cDNA by RT

2. DNA fragments placed on a chip

3. RNA's for comparison can be colored coded
color indicates relative levels of gene expression in two sources of mRNA
Describe gene inactivation methods using RNAi.
-antisense RNA can bind and inactivate mRNA

-siRNA or RNAi; short interfering RNA, double stranded RNA that can cause mRNA degradation

-RNAi, can block translation

-RNAi, can cause chromatin (DNA) to form an inactive condensed structure
Describe how PCR can be used diagnostically for Huntinton's disease.
-Defect results from an amplification of a trinucleotide (CAG)n within the gene.

-Primers on either side of the repeat can be used to amplify the region and the size (number of times CAG is repeated) determined.

-PCR trechnique now allows unambigious genetic screening.
What was the result of experiments where neurons transformed by a gene containing the CAG repeats of Huntington's disease coupled with green fluorescent protein (GFP) with studies by microscopy?
-cells with inclusion bodies have lower diffuse fluorescence and are less likely to die

-suggests a precursor to the inclusion body is the toxic factor

-N-terminal fragment of Htt may cause disease

-cause of disease may be through Htt binding trancription factors

-may block histone acetylase, enzyme that helps turn on gene expression

-Htt causes increased Ca2+ transport, causing mitochondria to turn on apoptosis

-Htt may reduce transport of signals that stabilize neurons (brain derived neurotrophic factor)

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