Chapter 17 BioTechnology

A molecular biology lab workflow

• Extract DNA or RNA from cells.

• Amplify a target region with PCR.

• Cut DNA with restriction enzymes when needed.

• Separate fragments by gel electrophoresis and interpret the pattern.

EXAM Q: Which sequence best represents the laboratory workflow emphasized in chapter 17

Extraction → PCR → restriction digest → gel electrophoresis

DNA and RNA Extraction

• Lysis buffer (mostly detergent) breaks

down lipids in the cell and its

membranes.

• After the cells are broken down, they are

treated with enzyme proteases to

further break down material.

• Remaining material is centrifuged to

separate it.

• The supernatant (liquid) containing the

DNA/RNA is extracted.

PCR/Gel Electrophoresis

• Polymerase chain reaction, or PCR, is used to amplify a

specific DNA sequence

• Primers short complementary DNA, genomic DNA, Taq

polymerase, and deoxynucleotides.

• DNA can be separated on the basis of size using gel

electrophoresis.

• DNA has a negative charge, moves towards a positive

pole

• Agarose gel acts as a sieve sorting fragments by size.

• Small fragments move more faster

EXAM Q: Select ALL statements that correctly describe PCR or agarose gel electrophoresis

DNA moves toward the positive electrode during gel electrophoresis because DNA is negatively charged.

Smaller DNA fragments generally move farther through an agarose gel than larger fragments.

Gel electrophoresis copies DNA molecules so that they can be detected.

PCR is used to make many copies of a specific DNA region

Restriction enzymes cut specific DNA sequences (slide)

• Restriction enzymes are bacterial enzymes that recognize specific DNA sequences

• They cut both strands of DNA at predictable postions

• Each enzyme has its own recognition sequence

• Cutting produces DNA pieces called restriction fragments

• Restriction enzymes are bacterial enzymes that recognize ____ ____ ____

specific DNA sequences

Restriction enzymes _____ both strands of DNA at predictable positions

cut

Cutting produces DNA pieces is called

restriction fragments

Sticky ends vs blunt ends (slide)

Many recognition sites are palindromic when both strands are read 5′→3′.

• Staggered cuts leave single-stranded

overhangs called sticky ends.

• Straight cuts across both strands

produce blunt ends.

• Sticky ends can base-pair with

complementary DNA ends.

DNA cutting: Many recognition sites are _______ when both strands are read 5′→3′.

Palindromic

Staggered DNA cuts leave single-stranded

overhangs called ___ _____

sticky ends

Straight cuts across both DNA strands produce _____ _____

blunt ends

_____ _____ can base-pair with complementary DNA ends.

sticky ends

Joining DNA with compatible sticky ends (slide)

DNA cut with the same restriction enzyme has compatible overhangs.

• Complementary sticky ends can anneal by base pairing.

• DNA ligase seals the sugar-phosphate backbone.

• This is the basis of recombinant DNA construction.

Joining DNA with compatible sticky ends: DNA cut with the same restriction enzyme has _______ _______

compatible overhangs

Complementary sticky ends can ______ by base pairing

anneal

Joining DNA with compatible sticky ends: ___ _______ seals the sugar phosphate backbone

DNA Ligase

Lab workflow: PCR → restriction digest → gel

PCR makes enough copies of the target DNA to analyze.

• A restriction enzyme tests whether its recognition site is present in the PCR product.

• Digestion changes fragment sizes when the recognition site is cut.

• Gel electrophoresis reveals the resulting band pattern.

PCR makes enough copies of the ____ _____ to analyze.

Target DNA

A restriction enzyme tests ….

whether its recognition site is present in the PCR product.

• Digestion _____ _____ _____ when the recognition site is cut.

changes fragment sizes

Gel electrophoresis reveals the ______ _____ ____

resulting band patterns

Reading a restriction digest (slide)

An uncut PCR product appears as one band.

• A cut product appears as two smaller bands.

• If cut and uncut versions are both present, all corresponding bands can appear.

• Compare band positions with the DNA ladder to estimate fragment sizes.

Reading a restriction digest: An uncut PCR product appears as ____ ____

one band

Reading a restriction digest: A cut product appears as ____ ____ ____

two smaller bands

Reading a restriction digest: If cut and uncut versions are both present, ____ _______ _____ ____ ____

all corresponding bands can appear

Reading a restriction digest: To estimate fragment sizes, ….

Compare band positions with the DNA ladder

Blotting: targeted detection (slide)

Southern blot: DNA fragments detected

with a labeled nucleic-acid probe.

• Northern blot: RNA detected with a

labeled nucleic-acid probe.

• Western blot: proteins detected with a

specific antibody.

• The general idea is separaƟon → transfer

to a membrane → targeted detecƟon.

Southern Blot

DNA fragments detected with a labeled nucleic-acid probe.

Northern Blot

RNA detected with a labeled nucleic-acid probe.

Western blot

Proteins detected with a specific antibody

The general idea of blotting is…

seperation → transfer to a membrane → targeted detection