Text Notes BIO130 Lab 2

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9 Feb 2012
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Lab 2 Text Notes (pg. 532-534, 540-541, 534)
Analyzing and Manipulating Data
- cleavage of DNA at specific sites by restriction nucleases – allows for isolation
and manipulation of individual genes
- DNA ligation – design and construct DNA molecules not in nature
- DNA cloning – use of cloning vectors or polymerase chain reaction (portion of
DNA repeatedly copied – generates billions of identical molecules)
- nucleic acid hybridization – can find specific DNA/RNA sequence of basis of its
ability to selectively bind complementary nucleic acid sequences
- determine sequence of DNA nucleotides – identify genes, deduce amino acid
sequence of proteins they encode
- monitor levels of mRNA produced by every gene in a cell with nucleic acid
arrays – tens of thousands of hybridization reactions take place
simultaneously
Restriction Nucleases
- gene – not a discrete entity – small region of much longer DNA molecule
- DNA molecules cannot be easily separated (because they consist of
approximately the same mixture of the same four nucleotides) on the basis of
different charges and binding properties
- restriction nucleases – enzymes purified from bacteria – cleave long strands
of DNA double helices at specific site into fragments of defined sizes
- restriction nucleases each have different sequence specificities
- an enzyme can create a DNA fragment with a particular gene – fragment size
is used as a basis for partial purification of the gene from the mixture
- different bacteria = different nucleases – protects from viruses through
degradation of viral DNA
- bacterial nuclease recognizes specific sequence (4-8 nucleotides) – protected
from cleavage by methylation at A or C nucleotide – sequences in foreign
DNA normally not methylated – cleaved by restriction nucleases
- some restriction nucleases produce staggered cuts – short single stranded
tails at two ends of each fragment – cohesive ends – tail can form
complementary base pairs with tail of any other end produced by the same
enzyme – two DNA fragments can be joined together (as long as fragments
are generated with some restriction nuclease or one producing the same
cohesive ends)
- DNA molecules produced through splicing two DNA fragments together
recombinant DNA molecules
DNA Separation by Gel Electrophoresis
- electrophoresis used in analysis of proteins – can also determine length and
purity of DNA molecules
- nucleotide in nucleic acid molecule carries single negative charge (phosphate
group) therefore does not need to add charged detergent (SDS)
- specific polyacrylamide gels – allow separation of molecules of different
lengths by even a single nucleotide – pores do not allow large DNA molecules
to pass however
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