Exam 2: Cloning / Biotechnology

Recombinant DNA technology

Set of techniques used to locate, isolate, alter, and study DNA segments.

Combine DNA from two distinct sources

What is the major goal of recombinant DNA technology?

Restriction endonuclease

What is another name for a restriction enzyme?

Specific nucleotide sequences in DNA

What do restriction enzymes recognize before cutting DNA?

Double-stranded cuts

What type of cuts do restriction enzymes make in DNA?

Restriction site

What is the specific DNA sequence where a restriction enzyme cuts?

Bacterial defense against viruses

What is the natural biological function of restriction enzymes?

Type II restriction enzymes

Which restriction enzyme class is most commonly used in molecular genetics?

Bacterial species of origin

What does the name of a restriction enzyme generally indicate?

EcoRI

What classic restriction enzyme name indicates origin from Escherichia coli?

4–8 base pairs

Approximately how long are restriction-enzyme recognition sequences?

Palindromic restriction site

A recognition sequence that reads the same 5′→3′ on both complementary strands.

Same 5′→3′ sequence on both complementary strands

★ HIGH YIELD — What does palindromic mean for a restriction site?

Cohesive ends

Restriction-fragment ends produced by a staggered DNA cut.

Sticky ends

What is another name for cohesive ends?

Single-stranded overhangs

What structural feature is produced by a staggered restriction-enzyme cut?

Complementary overhangs can spontaneously base-pair

Why are cohesive ends useful for joining DNA fragments?

Blunt ends

Restriction-fragment ends produced by an even cut through both DNA strands.

No single-stranded overhang

What distinguishes a blunt end from a cohesive end?

Staggered cut → cohesive/sticky end

★ HIGH YIELD — What type of restriction cut produces cohesive ends?

Even cut → blunt end

★ HIGH YIELD — What type of restriction cut produces blunt ends?

Bacteriophage T4 ligase

What ligase is specifically noted for joining blunt-ended fragments?

Restriction enzyme = cut DNA at a specific sequence

★ HIGH YIELD — What is the primary function of restriction enzymes in biotechnology?

Gene cloning

Amplifying a specific piece of DNA using a bacterial cell.

Foreign DNA introduced into replicating cells

How does gene cloning generate many identical copies of a DNA fragment?

Restriction enzyme cleavage

What is the first major step in cloning DNA?

Many DNA fragments

What does restriction-enzyme digestion initially create from the source DNA?

Cloning vector

A DNA molecule used to carry and replicate a foreign DNA fragment in a host cell.

Hybrid/recombinant molecule

What is a cloning vector containing an inserted foreign DNA fragment called?

Single bacterial host cell

Into what is the recombinant cloning vector introduced for amplification?

Clone

A population derived from one host cell in which each bacterium carries copies of the same inserted DNA.

Restriction enzymes

How can the amplified cloned DNA fragment later be released from the vector?

Autonomous replication

★ HIGH YIELD — What replication property must a cloning vector possess?

Selectable marker

★ HIGH YIELD — What feature allows identification/selection of cells carrying a cloning vector?

Antibiotic-resistance gene

What common example of a selectable marker is given?

Restriction-enzyme recognition site

What DNA feature must a cloning vector contain to allow foreign DNA insertion?

Replication + selectable marker + restriction site

★ HIGH YIELD — What three major properties must a cloning vector have?

Plasmids and viruses

What are the two most common cloning vectors listed?

Bacterial plasmid

A small circular extrachromosomal DNA molecule found in bacteria.

Extrachromosomal DNA

What does it mean that a plasmid exists separately from the bacterial chromosome?

Antibiotic resistance

What example of a “special” gene carried by plasmids is given?

Transfer between bacterial cells

What natural behavior of plasmids is mentioned in the lecture?

Easy isolation from bacterial cells

What practical feature makes plasmids useful as vectors?

Transformation

★ HIGH YIELD — What is the process of introducing foreign DNA into a bacterial or yeast cell?

Transformation = DNA enters the host; cloning = DNA is amplified

★ HIGH YIELD — Distinguish transformation from cloning.

Restriction enzyme

★ HIGH YIELD — Which enzyme cuts donor DNA and vector DNA?

DNA ligase

★ HIGH YIELD — Which enzyme joins the foreign DNA fragment to the vector?

Cut → insert/join → transform → replicate

★ HIGH YIELD — What is the basic order of DNA cloning?

DNA library

A collection of clones containing all DNA fragments from one source.

Genomic DNA library

A library produced from total genomic DNA cut with restriction enzymes and ligated into vectors.

Total DNA

★ HIGH YIELD — What source material is represented in a genomic DNA library?

cDNA library

A DNA library containing only sequences that were transcribed into mRNA.

Expressed genes / mRNA-derived sequences

★ HIGH YIELD — What does a cDNA library represent?

Genomic library = all DNA; cDNA library = transcribed/mRNA-derived DNA

★ HIGH YIELD — Contrast genomic and cDNA libraries.

Reverse transcriptase

Which enzyme is shown converting mRNA into cDNA for a cDNA library?

mRNA → cDNA

What is the starting nucleic-acid conversion used to construct a cDNA library?

Sanger dideoxy sequencing

A classic method used to determine the base sequence of a cloned DNA fragment.

Single-stranded DNA template

What type of DNA template is used in the classic Sanger method?

Radiolabeled primer

What is added complementary to the 3′ end of the target DNA in Sanger sequencing?

dATP, dTTP, dCTP, dGTP

Which four normal deoxynucleotide triphosphates are included in Sanger sequencing?

ddNTP

Dideoxynucleotide that terminates DNA-chain elongation during Sanger sequencing.

No 3′ hydroxyl group

★ HIGH YIELD — What key structural feature is missing from a ddNTP?

Chain termination

★ HIGH YIELD — What happens when a ddNTP is incorporated into the growing DNA strand?

Different-length DNA fragments

What does Sanger sequencing generate because ddNTPs terminate synthesis at different positions?

ddNTP lacks 3′-OH → elongation stops

★ HIGH YIELD — What is the core mechanism of Sanger chain termination?

Southern blotting

A technique used to detect mutations or DNA sequences of interest.

DNA

★ HIGH YIELD — What type of molecule is analyzed by Southern blotting?

Restriction enzymes, electrophoresis, and a DNA probe

What major tools are combined in Southern blotting?

Extract DNA → cut → electrophoresis → denature → blot → probe

★ HIGH YIELD — What is the basic Southern-blot sequence?

Restriction enzymes

What is used to cut DNA before electrophoresis in a Southern blot?

Electrophoresis

What technique separates the DNA fragments in a Southern blot?

Denaturation

What step separates the two DNA strands before probing in a Southern blot?

Membrane

Onto what surface is the separated DNA transferred in Southern blotting?

Complementary DNA probe

What binds the DNA sequence of interest in a Southern blot?

Hybridization

What process allows the complementary probe to bind its target DNA?

PCR

Polymerase chain reaction.

Test-tube amplification of selected DNA

★ HIGH YIELD — What is the purpose of PCR?

Specific primers

What determines which DNA region is amplified during PCR?

Thermostable DNA polymerase

What kind of polymerase is required for PCR?

Exponential amplification

How does the amount of target DNA increase over repeated PCR cycles?

Each cycle doubles the target DNA

What happens to DNA quantity during each ideal PCR cycle?

Denaturation → annealing → extension

★ HIGH YIELD — What is the correct order of PCR steps?

Denaturation

Which PCR step separates the two DNA strands?

Approximately 90–100°C

What temperature range is shown for PCR denaturation?

Annealing

Which PCR step allows primers to bind complementary target sequences?

Approximately 30–65°C

What temperature range is shown for PCR primer annealing?

Extension

Which PCR step uses DNA polymerase to synthesize new DNA?

Approximately 60–70°C

What temperature range is shown for PCR extension?

Separate → primers bind → synthesize

★ HIGH YIELD — What simple description corresponds to denaturation, annealing, and extension?

Thermostability

Why must the DNA polymerase used in PCR withstand high temperature?

Gel electrophoresis, Southern blotting, or direct sequencing

How can amplified PCR products be analyzed according to the lecture?

PCR = test-tube amplification; cloning = amplification using replicating host cells

★ HIGH YIELD — Contrast PCR with gene cloning.

Northern blot

A technique used to analyze specific mRNA molecules.

mRNA / RNA

★ HIGH YIELD — What type of molecule is analyzed by a Northern blot?

Electrophoresis → membrane transfer → labeled probe

What is the basic workflow of Northern blotting?

Radiolabeled probe complementary to target mRNA

What detects the RNA of interest in a Northern blot?

Band strength

What Northern-blot feature reflects the amount of target mRNA?

Band position

What Northern-blot feature reflects the size of the target mRNA?

Amount and size of mRNA

★ HIGH YIELD — What two things can a Northern blot reveal about a particular mRNA?

Microarray

A gene-analysis method containing thousands of immobilized DNA sequences.