Cell Biology Lecture 32: Biotechnology

(DNA Manipulation) What is the method used to isolate DNA?

Cell Lysis

(DNA Manipulation) What is the method used to quantitate DNA?

UV Absorbance

(DNA Manipulation) What is the method used to detect DNA?

Agarose Gel Electrophoresis

(DNA Isolation) What is the method used to isolate plasmid DNA?

Alkaline Lysis

(DNA Isolation) What are the steps of alkaline lysis?

1. Lyse cells with alkali

2. Neutralize

3. Centrifuge

4. Alcohol + salt precipitate

5. Re-suspend DNA

(DNA Quantitating) What is the absorption maximum of nitrogenous bases in nucleotides?

260 nm

(DNA Quantitating) What is the absorption maximum of proteins?

280 nm

(DNA Quantitating) Why do proteins have a higher UV absorption maximum?

Tryptophan residues

(DNA Detection) What trait of DNA allows agarose gel electrophoresis to work?

Net negative charge

(DNA Detection) Where do DNA migrate towards during agarose gel electrophoresis?

Towards the anode (positive electrode)

(DNA Detection) What is DNA forced to move through in agarose gel electrophoresis?

Molecular proportions made of agarose

(DNA Detection) Do larger or smaller pieces move slower in agarose gel electrophoresis?

Larger

(DNA Detection) How can DNA be visualized in agarose gel electrophoresis?

Staining with ethidium bromide + UV light

(DNA Mapping/Cleaving) What is the general use of restricting nucleases on substrates?

1. Slice off pieces of ends

2. Cut middle of substrate

(DNA Mapping/Cleaving) What is the substrate of restriction enzymes?

Double-stranded DNA

(DNA Mapping/Cleaving) Are restriction enzymes endonucleases or exonucleases?

Endonucleases

(DNA Mapping/Cleaving) What sequence is the result of restriction enzyme cutting of double stranded DNA?

Palindrome Sequence

(DNA Mapping/Cleaving) What ends are the results of restriction enzyme cleaving?

1. Blunt Ends

2. Sticky Ends

(DNA Mapping/Cleaving) What are restriction enzymes named after?

Bacterium

(DNA Mapping/Cleaving) What is a restriction map?

Linear array of sites for restriction enzymes for any given DNA

(DNA Mapping/Cleaving) What protects host DNA from restriction enzymes?

Host DNA Methylation

(DNA Mapping/Cleaving) What can be used to map genes?

Restriction enzymes

(DNA Mapping/Cleaving) What results from varying distance between restriction sites (length of fragments)?

Restriction Fragment Length Polymorphism (RFLP)

(DNA Mapping/Cleaving) What causes varying distance between restriction sites?

Unique chromosome sequence between individuals

(DNA Mapping/Cleaving) What can RFLPs be used for?

Genetically tell individuals apart

(DNA Mapping/Cleaving) Why can RFLPs show relationships between individuals?

Children inherit genetic elements

(DNA Mapping/Cleaving) What can be used for DNA cloning?

Restriction Enzyme

(DNA Hybridization) What method is used to identify DNA fragments that contain specific sequences?

Southern Blots

(DNA Hybridization) What is the DNA fragment used in Southern Blotting with a specific known sequence that is labeled with a detectable tag?

DNA probe

(DNA Hybridization) What will DNA probes only bind to?

Sequences that are highly similar to its own sequence

(DNA Hybridization) What can identify sequences that are present/absent in complex mix of DNA molecules?

DNA Microarrays

(DNA Hybridization) How many genes are monitored by hybridization on DNA microarray?

Thousands at once

(DNA Hybridization) What are the steps of Southern Blotting?

1. Gel Electrophoresis

2. Blot DNA fragments onto nitrocellulose paper

3. Remove nitrocellulose paper

4. Hybridization of DNA probe to separate DNA

5. Labeled DNA probe visualized by autoradiography

(DNA Hybridization) What are the uses of Southern Blotting?

1. Identify restriction fragments

2. Locate/map genes on chromosome

3. Determine number of copies

4. Identify positive clones

(DNA Hybridization) What are DNA microarrays used for?

Thousands of different genes are immobilized

(DNA Hybridization) What does the order of sequences on microarrays allow the analysis of?

Functional Analyses

(DNA Hybridization) What can be determined by DNA microarrays?

Expression of thousands of genes

(DNA Amplification) What is used for DNA amplification?

Polymerase Chain Reaction (PCR)

(DNA Amplification) What is the machine that performs PCR?

Thermocycler Machine

(DNA Amplification) What are the requirements for PCR?

1. Primers + primer binding site sequence

2. Heat-stable DNA polymerase

(DNA Amplification) What acts as primers for PCR?

Two sequence specific oligo nucleotides

(DNA Amplification) Can PCR amplify antibodies?

No

(DNA Amplification) What are the final products of PCR?

DNA fragments with sequences bracket by the primers

(DNA Amplification) What are the applications of PCR?

1. Amplifies a specific DNA sequence

2. RT-PCR (mRNA Level)

(DNA Sequencing) What method is used for DNA sequencing?

Dideoxy-based sequencing method

(DNA Sequencing) How does the dideoxy-based sequencing method work?

Prevents strand extension at 3' end

(DNA Sequencing) What does the dideoxy-based sequencing method determine?

Sequence of fragment of DNA or gene

(DNA Sequencing) What does the dideoxy-based sequencing method detect?

Nucleotide mutations

(RNA Manipulation) What is the only difference between DNA and RNA isolation?

Measures to exclude RNases

(RNA Manipulation) Are many RNases extremely stable?

Yes

(RNA Manipulation) What makes isolating RNA technically demanding?

Presence of RNases

(Protein Manipulation) What conditions must the isolation of proteins be done under?

Conditions that inhibit proteases

(Protein Manipulation) What should not be disrupted during protein isolation?

Higher structures

(RNA & Protein Quantitating) What ratio is used as a criterion of purity of protein/nucleic acid samples?

A280/A260

(mRNA Levels) What is used to generate cDNA from mRNA?

Reverse transcriptase

(mRNA Levels) What techniques can be used to analyize mRNA levels using cDNA?

1. RT-PCR

2. Micro-array analysis

3. RNA-seq

(mRNA Levels) What is RT-PCR used to determine?

Precise amount of starting mRNA template qRT-PCR

(mRNA Levels) What is micro-array analysis used to determine?

Precise amounts of mRNA immobilized on an array

(mRNA Levels) What is RNA-seq used to determine?

Total mRNAs

(Protein Analysis) Which technique is used to separate proteins based on molecular weight?

SDS-PAGE

(Protein Analysis) Which technique is used to separate proteins based on molecular weight and pI?

2-Dimensional Gel Electrophoresis

(Protein Analysis) What techniques are used in 2-Dimensional Gel Electrophoresis?

1. Isoelectric point gel

2. SDS-PAGE

(Protein Analysis) What are Western Blots used to detect?

Specific protein using a probe

(Protein Analysis) What technique uses antibodies to detect presence of antigens?

Enzyme Immunoassays (EIAs)

(Protein Analysis) What are the different formats of antigen to surface binding?

1. Immunostaining

2. ELISA

3. Immunochromatographic assays

(Protein Analysis) What is used to stain in immunostaining?

Enzyme-linked antibodies

(Protein Analysis) Where does the enzyme bind in ELISA?

Constant region of antibody

(Protein Analysis) What are immunichromatographic assays also called?

1. Lateral Flow Tests

2. Strip Tests