special DNA "hook" ; scientists need this when searching for a particular piece of DNA

a probe is usually a form of DNA called single-stranded DNA (half a DNA- one backbone with bases sticking out from it). If a piece of single-stranded DNA finds another piece of single-stranded DNA with the matching base sequence, it will pair with it to make a double stranded DNA helix. This pairing of two single DNA strands to make a double helix is Hybridization

Combination of restriction analysis, transfer to membrane, and hybridization to prove

it is important if we need to have information about which region of the sample DNA molecule the sequence of interest is located.

this enzyme cuts the DNA sequence 5'CCCGGG3' between the C and G in the middle of the sequence

specific for their targets because these enzymes recognize specific DNA sequences and then cut the DNA within the recognition sequence.

Restriction Fragment Length Polymorphism (RFLP) or DNA fingerprinting

How would you use RFLP to analyze a crime scene?

What are the three steps in each cycle of PCR and what is happening in each step?

1. denaturation- separation of template DNA strands (PCR uses high temperature to separate the strands, or melt the DNA duplex)
2. annealing- hybridization of the primer to the template (temperature decreased to allow the primers to form hydrogen bonds since polymerase will only extend a primer that is hydrogen bonded to the template)
3. extension- DNA synthesis of the complementary strand (DNA polymerase can now read the template and add free nucleotides that are complementary to the template onto the 3' end of the primer)

What components are required for a PCR reaction?

DNA template

two primers

enzyme DNA polymerase enxyme

free nucleotides

similarites:

• they both copy DNA
• they both initially have to separate the double stranded DNA (double helix)

differences:

• PCR makes many multiple copies of DNA while replication makes one copy of DNA
• PCR is a scientific procedure; while replication occurs automatically in nature

to analyze your PCR results we need to determine the size of the PCR products

aka DNA standards

these DNA markers contain fragments of known size and can be purchased continaing various sizes of DNA fragments depending on the size you expect your product to be.

What are the reaction requirements for transcription (in vivo)? What is the product?

promoter

an RNA polymerase

a template

and ribonucleoside triphosphates

product: RNA

mRNA containing a ribosome-binding site and coding region

ribosomes

transfer RNAs (tRNAs)

amino acids

tRNA synthetases to attach the amino acids to the tRNAs

product: protein

initiation

elongation

termination

prokaryotes: transcription and translation (gene expression) is coupled

eukaryotes: gene expression not coupled (diff compartments. transcription nucleus. translation cytosol.)

Polyacrylamide Gel Electrophoresis. a technique that can be used to separate a mixture of macromolecules (such as polypeptides or nucleic acids), to determine the size of a biological molecule, or to identify the molecule.

completely denatures proteins so that they can be separated based solely on size (Molecular weight)

-charged molecules migrate toward the oppositely charge pole when placed in an electric field. This technique can be used to separate molecules of different sizes as they move through the pores of a continuous gel matrix.

-proteins are denatured and kept from precipitating by boiling them in the presence of a reducing agent, like BME. detergent, like SDS.

-Reducing agent breaks the covalent disulfide bonds, while heating the protein helps break weaker bonds (ionic bonds and hydrogen bonds)

-SDS helps disrupt hydrophobic interactions in the protein (because it is an amphiphathic molecules- containts both hydrophobic[long hydrocarbon chain] and hydrophilic[negative charged sulfate group] region)

-The hydrophobic tails of SDS disrupt the weak hydrophobic interactions of the protein that are involved in maintaing secondary and tertiary structure. The hydrophobic tails then remain associated with the nonpolar amino acids in the polypeptide so that the hydrophilic heads extend away from the protein into the surrounding aqueous solution. The result is a protein that has unfolded into a linear polypeptide with a coating of negative charges. The negatice charge conferred by the SDS overwhelms any inherent charge that would normally be found on the surface of the protein. Thus all of the denatured proteins have a net negative charge and migrate toward the positive pole in an electric field.

a method for amplifying a region of DNA by repeated cycles of DNA synthesis in vitro

if no DNA in the sample can hybridize to the primers, no PCR product will be synthesized

What is anneal?

What is coupled?

to form hydrogen bonds with

can occur at the same time.

a technique that can be used to separate a mixture of macromolecules to determine the size of a biological molecule, or to identify the molecule. Pores are formed by creating a latticework with a chemical known as acrylamide.

the type of PAGE used in lab completely denatures proteins so that they can be separated based solely on molecular weight.

1. the reducing agent (BME) breaks down the covalent disulfide bonds
2. the detergent (SDS) helps to disrupt hydrophobic interactions in the protein.
3. the heat helps to break down the weaker bonds such as ionic bonds and covalent bonds