Biochemistry 2280A Chapter Notes - Chapter 10: Dna Microarray, In Situ Hybridization, Fmr1

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Biochemistry 2280 – Topic 21 – Recombinant DNA Technology Tools
Textbook Notes
Ch. 10 – Modern Recombinant DNA Technology
Manipulating and Analyzing DNA Molecules
RESTRICTION NUCLEASES CUT DNA MOLECULES AT SPECIFIC SITES
Restriction nuclease: enzyme that degrades foreign DNA in bacteria by cleaving it at
specific nucleotide sequences
oThose sequences are chemically modified in bacteria’s own DNA to avoid
cleaving
oTarget sequences are palindromes – 4-8 nucleotides long
oCreates either blunt cuts or overhangs (sticky ends)
oVery useful in genetic engineering
oEnzymes with longer target sequences generally produce longer fragments
GEL ELECTROPHORESIS SEPARATES DNA FRAGMENTS OF DIFFERENT SIZES
Urea added to gel to ensure single stranded DNA remains single stranded
BANDS OF DNA IN GEL CAN BE VISUALIZED USING FLUORESCENT DYES OR
RADIOISOTOPES
Expose agarose or polyacrylamide gel to fluorescent dye that can show bands when
seen under UV light
More sensitive detection – incorporate 32P into DNA before gel electrophoresis
HYBRIDIZATION PROVIDES SENSITIVE WAY TO DETECT SPECIFIC NUCLEOTIDE
SEQUENCES
Hybridization: reformation of hydrogen bonds between complementary base pairs (DNA
renaturation)
Southern blotting method uses small, single-stranded DNA probes (with fluorescent or
radioactive tag to reveal DNA bands with sequence of interest
DNA Cloning in Bacteria
DNA CLONING BEGINS WITH GENOME FRAGMENTATION AND PRODUCTION OF
RECOMBINANT DNAs
Fragments joined together by DNA ligase to produce recombinant molecules not found
in nature
RECOMBINANT DNA CAN BE INSERTED INTO PLASMID VECTORS
Plasmids have cleavage sites for common restriction nucleases, therefore can be
conveniently opened and foreign DNA can be inserted
RECOMBINANT DNA CAN BE COPIED INSIDE BACTERIAL CELLS
Transformation: natural uptake of plasmids by bacteria
In test tube, E. coli cells coaxed to take up recombinant plasmids, then allowed to grow
in nutrient-rich broth
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Plasmid contains resistance against specific antibiotic, add antibiotic to tube so that only
bacteria that have taken up plasmid can survive
Bacteria then lysed, plasmid purified from other cell contents
DNA fragments recovered by using same restriction nuclease used to insert it, then
separated with gel electrophoresis
Fragment has been successfully amplified
GENES CAN BE ISOLATED FROM DNA LIBRARY
In reality, restriction nuclease doesn’t just create one fragment but many fragments
All fragments inserted into vectors, then bacterial cells with recombinant plasmid of
interest isolated
DNA library: collection of cloned DNA fragments in bacterial culture
oIf recombinant fragments derived from chromosomal DNA from organism of
interest, collection called genomic library
DNA probes, first created by applying genetic code in reverse, used to bind to and find
particular gene in library
Now we have electronic database, DNA libraries are unnecessary
cDNA LIBRARIES REPRESENT mRNAs PRODUCED BY PARTICULAR CELLS
To amplify eukaryotic protein-coding DNA in bacterial cells, we need version with exons
only
cDNA library: collection of DNA prepared by reverse transcription of extracted mRNA
ocDNA = complementary DNA
ocDNA doesn’t contain noncoding, regulatory, spacer, and intron sequences
cDNA clones sued to study how gene expression varies between stages of development
and other cellular conditions
DNA Cloning by PCR
Polymerase chain reaction (PCR) is fast way to clone DNA
oinvented in 1980s
odoesn’t rely on bacteria, all done quickly in test tube
oonly single copy of DNA needed to start (can be from haploid sperm, for
example)
PCR USES DNA POLYMERASE TO AMPLIFY SELECTED DNA SEQUENCES IN TEST TUBE
Primers used in PCR not only to guide polymerase but also direct it to specific DNA
sequence to be amplified
MULTIPLE CYCLES OF AMPLIFICATION IN VITRO GENERATE BILLIONS OF COPIES OF
DESIRED NUCLEOTIDE SEQUENCE
PCR is method of choice for cloning fragments under 10 000 nucleotides
Billions of copies made in few hours
Original template can be DNA or RNA, no need to create library first
oRNA will be first converted into cDNA
PCR IS ALSO USED FOR DIAGNOSTIC AND FORENSIC APPLICATIONS
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