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Lecture 12

BIOL 3113 Lecture Notes - Lecture 12: Polymerase Chain Reaction, Molecular Cloning, Complementary Dna

Course Code
BIOL 3113
Barbara S

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12 DNA Technology
Collection of techniques to study DNA structure and function
*Cleavage of DNA at specific sites
enzymes - restriction nucleases
*DNA sequencing by gel electrophoresis
analysis of DNA base sequences
*Nucleic acid hybridization
based on complementary base pairing
uses labeled DNA or RNA to locate genes on DNA
*DNA cloning
copying of DNA sequences
polymerase chain reaction
*DNA engineering
modification of DNA sequences
insertion into cellular DNA
Cell culture - source of homogenous cells for studying genes, proteins, molecular
mechanisms, etc.
Cells can be isolated from tissues and grown in culture
*differentiated cells
maintain properties characteristic for cell type
have finite life time in culture
can be “immortalized” and propagated for long time in culture
*undifferentiated cells
can divide and grow indefinitely in culture
can be stimulated to differentiate to a different cell types
*malignant tumor cells
can be propagated indefinitely in culture
Restriction Nucleases
*Bacterial enzymes that cut (hydrolyze) DNA at specific sequences of 4-8 nucleotides
(restriction site)
*hundreds of different enzymes identified
Eco RI
Hind III
Not I
Alu 1

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Treatment of DNA molecule from the same species with a given restriction nuclease will
always produce the same set of DNA fragments
*Produce restriction fragments visible by gel electrophoresis
*Map of restriction (cutting) sites - restriction map
shows locations of restriction sites for one enzyme in relationship to restriction sites of
other enzymes
*restriction map - one of the early applications of restriction nucleases construction of
physical maps of small DNA regions
Ultimate physical map of DNA - complete nucleotide sequence of the genome
The existence of restriction nucleases makes DNA technology possible
DNA Sequencing - Dideoxy (Enzymatic) Method
More efficient (faster and quicker) way of determining the nucleotide sequence
(developed in 1970s - still in use)
Uses four different dideoxyribonucleotides (ddATP, ddGTP, ddCTP, ddTTP) to
randomly terminate replication in vitro
Dideoxyribonucleotides lack 3’ hydroxyl group
Dideoxy or Enzymatic Method
*Produces collection of different DNA copies that terminate at every position in the
original DNA - different in length by a single nucleotide
*DNA copies separated on the basis of their length by PAGE
*Nucleotide sequence of the original DNA determined from the order of fragments in the
Now completely automated process
Robotic devices:
*mix reagents, load and run gels (chain terminating dideoxyribonucleotides tagged with
different fluorescent dies - allows all 4 synthesis reactions to be carried in the same tube
and run in one line of the gel)
*detector reads the nucleotide order from the gel sequences of analyzed fragments
recorded on the computer and complete sequence assembled from fragments
Speed limited only by size of the gel - maximum a few hundred nucleotides can be read
from a single gel

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Nucleic Acid Hybridization (Renaturation)
Method for locating genes within the genome - isolated or in situ
Based on fundamental property of DNA - formation of base pairs with complementary
nucleotide sequence (DNA/DNA, RNA/RNA, DNA/RNA)
*Denaturation of DNA double helix (90ºC or pH > 13)
*Mixing with labeled probe for a specific nucleotide sequence
*Renaturation (hybridization) at 65ºC
Single-stranded probes (10-1000 nucleotides long), commercially available or can be
synthesized in the laboratory. Method widely used in research and medicine
Over 3,000 genetic diseases are caused by mutation in single genes
Southern blotting - common laboratory procedure used to visualize the hybridization
In situ hybridization - locates DNA base sequences on chromosomes, within cells and
tissues, reveals the distribution of RNA in cells
Probes: DNA & RNA
Labels: Radioactive or Fluorescent
Hybridization on DNA microarrays - locates expression of multiple genes
New technique (developed in 1990s), currently one of the most important uses of nucleic
acid hybridization
Allows determining exactly:
*which genes are being transcribed into mRNA
*which are silent
during cell differentiation, life and neoplastic transformation, and in response to different
factors (growth factors, toxins, hormones, infection)
DNA fragments corresponding to entire gene or short synthetic nucleotides placed on
microarray slide, hybridized with cDNA (DNA complementary to mRNA extracted from
cells) labeled with fluorescent probe
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