LIFESCI 7A Lecture Notes - Lecture 8: Recombinant Dna, Restriction Enzyme, Restriction Site
WEEK 8
12.3: Isolation, Identification, and Sequencing of DNA Fragments
● The polymerase chain reaction selectively amplifies regions of DNA.
○ polymerase chain reaction (PCR) A selective and highly sensitive method for making copies
of a piece of DNA, which allows a targeted region of a DNA molecule to be replicated
(amplified) into as many copies as desired.
1. Denaturation: DNA heated/chemically treated to separate strands
2. Annealing: When solution cools, two primers anneal to complimentary sequence on
strands of duplex
3. Extension: Reaction heated to good temp for DNA polymerase to make new DNA
strands by extending primers in 5’ to 3’ direction
○ Ingredients required: Template DNA, DNA polymerase, all deoxyribonucleoside triphosphates
(A T C G), and two primers
○ Primer sequences are oligonucleotides
■ A short (typically 20 to 30 nucleotides), single-stranded molecule of known sequence
produced by chemical synthesis
○ Doubles number of DNA with each cycle
■ Target sequences appear after 3 cycles
○ Taq polymerase used (able to withstand high heat needed to denature DNA)
● Electrophoresis separates DNA fragments by size.
○ DNA is negatively charged (ionized phosphates in backbone), move to positive pole of
apparatus
○ Can be used to test effectiveness of PCR process
● Restriction enzymes cleave DNA at particular short sequences.
○ Cutting DNA used in recombinant tech, determining presence of specific sequences, breaking
up genome into smaller pieces to be analyzed
○ restriction enzyme t recognizes specific, short nucleotide sequences in double-stranded DNA
and cleaves DNA at or near these sites.
■ restriction site A recognition sequence in DNA cutting,
which is typically 4-6 base pairs longmost restriction enzymes
cleave double-stranded DNA at or near these restriction
sites.
■ Often palindromic (reads the same in
both directions on both strands)
■ Can leave 5’ overhang, 3’ overhang
(sticky ends), or no overhang
● DNA strands can be separated and brought back
together again.
○ renaturation (hybridization) The base pairing of complementary single-stranded nucleic acids
to form a duplex; opposite of denaturation.
■ Denatured DNA strands from one source can renature with DNA strands from a different
source if their sequences are precisely or mostly complementary
● More different the two strands, the cooler it needs to be to renature (this idea’s
used to measure how alike the strands are)
■ Used in comparing DNA between species (gene of one animal compared to same gene
of another)
■ Can be used to create probes by attaching a label to it and seeing which DNA
fragements it binds to
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