Cell and Molecular Biology Lecture 32: Biotechnology

What is the primary method explained for DNA isolation?

Lyse cells, separate DNA via DNA extraction

What is the method of choice to quantify pure DNA or pure protein?

UV absorbance

What is the primary example provided for DNA detection?

Agarose gel electrophoresis

What is the first step for DNA extraction?

Break down cell wall and membranes (physically)

What is the second step for DNA extraction?

Centrifuge to separate the solids from dissolved DNA

What is the third step for DNA extraction?

Precipitate DNA using isopropanol

What is the fourth step for DNA extraction?

Centrifuge a second time to separate DNA

What is the fifth step for DNA extraction?

Wash DNA pellet with ethanol and dry

What is the final step for DNA extraction?

Dissolve DNA

What method is employed for plasmid DNA isolation?

Alkaline lysis

What is the first step of alkaline lysis DNA extraction?

Lyse cells with alkali and detergents

What is the second step of alkaline lysis DNA extraction?

Neutralize sample to re-anneal plasmid

What is the third step of alkaline lysis DNA extraction?

Use centrifugation to remove denatured DNA, protein, and detergent, leaving dissolved plasmid behind

What is the fourth step of alkaline lysis DNA extraction?

Precipitate plasmid DNA using alcohol and salt

What is the final step of alkaline lysis DNA extraction?

Re-suspend the plasmid DNA in water or buffer

What is the absorption maximum of nitrogenous bases in nucleotides?

260 nm

What is the absorption maximum of proteins?

280 nm

What is the reason for the absorption maximum of proteins?

Tryptophan residues

What is the basic principle upon which agarose gel electrophoresis works?

DNA has a net negative charge

Which direction does DNA migrate?

Towards the anode

In agarose gel electrophoresis, larger pieces of DNA move __________ than small pieces of DNA

Slower

What is used for visualization of DNA on agarose gel?

Ethidium bromide + UV Light exposure

DNA ____________ can chew from the ends or attack the middle of the substrate

Nucleases

Restriction enzymes cut double-stranded DNA into short pieces in a sequence specific manner. What are these sequences called?

Palindrome sequences

What are the two types of ends that can be formed by RE activity?

Blunt ends or sticky ends

How are RE named?

After the bacterium from which they are isolated

What is the characteristic linear array of sites for restriction enzymes in any given DNA?

Restriction map

What is used to protect host DNA from restriction enzyme activity while still allowing foreign DNA to be digested?

DNA methylation of host DNA

What can be used to genetically tell individuals apart as well as show the genetic relationship between individuals?

Restriction fragment length polymorphism

What is used to identify DNA fragments that contain specific sequences?

Southern blots

What is the name for the specific sequence that is labeled with a detectable tag in southern blotting technique?

DNA probe

The probe used in southern blotting can only bind to what?

Identical or highly similar sequences (DNA/DNA or DNA/RNA)

What can be used to identify sequences that are present or absent in a complex mix of DNA molecules?

DNA microarrays

____________ on DNA microarray monitors the expression of thousands of genes at once

Hybridization

What is the first step of the Southern blot technique?

Electrophoresis of unlabeled DNA cut with RE

What is the second step of the Southern blot technique?

Separated DNA fragments blotted onto nitrocellulose paper

What is the third step of the Southern blot technique?

Nitrocellulose paper (with tightly bound DNA) is removed

What is the fourth step of the Southern blot technique?

Labeled DNA probe hybridized to separated DNA

What is the final step of the Southern blot technique?

Labeled DNA probe visualized by autoradiography

What is the Southern blot technique useful for?

Locate/map genes on chromosomes, determine number of copies of each gene in a genome, identify positive clones

What are solid supports onto which the sequences from thousands of different genes are immobilized at fixed locations?

DNA microarrays

What are the applications of PCR?

Exponential amplification of DNA, RT-PCR to study gene expression

PCR requires _______ sequence specific oligo-nucleotides to act as ________

Two, primers

What are the final products of PCR?

DNA fragments with sequences bracket by the primers

What is the foundational principle of dideoxy-based sequencing?

Using dideoxyribonucleoside prevents extension of strand at 3' end

How does dideoxy-based sequencing work?

DNA polymerase will add dideoxynucleotides, creating different length strands of DNA. These are sorted based on MW in a capillary tube gel and then imaged

RNA isolation is _______ to isolation of DNA

Similar

What is the difference between RNA and DNA isolation?

Careful measures taken to exclude RNases from the procedure

Why is it technically demanding to isolate RNA?

RNases are extremely stable and present everywhere

Why must isolation of proteins be done under specific conditions?

To inhibit proteases and preserve protein structure

What ratio is often used as a criterion of the purity of protein or nucleic acid samples during their purification?

A280/A260

What are the advantages of the ratio method for determining protein or nucleic acid sample purity?

Sample is not destroyed and it is very rapid

What are the techniques of using complementary DNA to analyze transcript levels?

RT-PCR, Microarray analysis, RNA-seq

RT-PCR can be used to generate cDNA and then followed by quantitative PCR to determine what?

The precise amount of starting mRNA template

Labeling cDNA with fluorescent dyes and using the fluorescent tags can be used for what?

Determining the precise amounts of multiple transcripts immobilized on an array

RNA-seq is used for what?

Deep sequence total mRNAs for individual mRNAs and amounts

SDS-Page separates proteins based on what?

Molecular weight (size)

2-dimensional gel electrophoresis separates proteins based on what?

Molecular weight (size) and pI (isoelectric point)

What comes first in 2-dimensional gel electrophoresis?

Isoelectric point gel

The western blot is similar to the southern blot, but it is used for ________ instead of DNA

Protein

The western blot is used to detect a _____________ using a __________

Specific protein, probe with a tag

The probe used in a western blot is an ___________ that specifically recognizes the protein of interest

Antibody

Enzyme Immunoassays use _______________ to detect the presence of _______________

Antibodies, antigens

What are the different formats of antigen binding to surface in enzyme immunoassays?

Immunostaining, ELISA, immunochromatographic assays, etc

What are immunochromatographic assays also known as?

Lateral flow tests or strip tests