Molecualr Biology Test 1

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Gel Mobility shift assay

Back 1

To check whether a protein will bind to a nucleic acid

Only need fragments

Bound DNA with protein will move slower on the gel

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Co-precipitation

Back 2

Protein-protein interaction

If antibody is available for YFP, it can be used to precipitate YFP via column

Epitope tag is available if an antibody is not present

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DNas foot printing

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-For DNA-protein interactions
-To determine where the protein interacts with nucleic acid
-Bound protein will hinder the digestion of DNA at binding sites
-DNA is end-labeled at one strand of DNA

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Cross-linking

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-Protein-protein interactions
-Irreversible reaction
-Inhibit enzyme activity
-e.g. glutaraldehyde reacts with primary amines to form an imine bond linking the protein to the glutaraldehyde

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SMRT

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Single molecule real time
-Example of 3rd (next) generation sequencing
-Base corporation release fluoresce close to the polymerase.
-Detect dNTP incorporation into single molecules based on fluorescence.

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Pyrosequencing or sequencing by synthesis (454 sequencing)

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-2nd generation sequencing
-Pely on coupled reaction to detect polymerization
-Release PPi to ATP, which in turn provides the energy for the luciferase to emit light

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Primer extension

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Using R.T. to extend a short primer to the 5' end of the RNA

Use this for "transcript mapping"

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In situ hybridization

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To locate genes on chromosomes or to detect expression in tissues

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Northern blot

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RNA on the membrane

Don't denature; RNA in NaOH RNA- RNA is chemically unstable i basic solutions

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Eukaryotic vector

Back 10

Used for protein production expression

Allow post-transcriptional modifications

Fold properly

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Shuttle vector

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Allow cloning and modification steps to be done in bacteria

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Phagemid

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Example- pBluescript

Replicate like a plasmid

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ORC

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Origin replication complex

DNA-binding complex that binds in all eukaryotes to origin of replication

Directs DNA replication

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qRT-PCR

Back 14

Quantitative reverse transcription PCR

qPCR

Called qRT-PCR if the template is cDNA

Requires specialized thermocycles to monitor
eg. SYBR green dye-intercalates btwn bases of dsDNA and fluoresces, so amt fluorescence indicated amt of product

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DNA A

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DNA/RNA hybrid or dsRNA; 11bp/turn

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DNA B

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Standard for of DNA; 10.5 bp/turn

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DNA Z

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dsDNA; left-handed and found in high [salt]

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AFLP

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Amplified fragment length polumorphism
-PCR based
-Use R.E. to digest gDNA. Ligase adapters to sticky ends of restriction fragments. Use primers complementary to adapter sequence.
-Restriction fragment is selecteded to be amplified.
-Amplified fragments are visualized on PAGE

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His tag

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His 6 (H6)
-Used to express YFP
-Make mRNA with YFP+EK+H6. Then translate and becomes protein with H6 tag along with different proteins. Then elute proteins in a column that has nickel or antibody for H6.
-Proteins with H6 tagged will be separated. Use EK to separate YFP and H6.

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Ontology

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Conceptualization of categories and/ or relationships between anything

Means a controlled vocabulary describing biochemical, cellular, or biological roles for every sequences.

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Polylinker

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aka Multiple cloning site (MCS)

Short segment of DNA that contains several continuous restriction sites, at which you place your insert

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RRM

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RNA recognition motif

Protein domain that consists of 4-beta sheets

Recognize sugar-phosphate backbone, intercalates with bases

Sequence specific

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Isoschizomer

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Restriction enzymes from different organisms with the same recognition sequence

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ChIP

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Chromatin immunoprecipitation

To investigate protein-DNA interactions; aimed at determining whether specific proteins are associated with specific genomic regions

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Cosmid

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(Cos sequence + plasmid)

A hybrid plasmid that contains lambda phage cos sequences; they can be useful to build genetic libraries, especially involving large inserts

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Zn motif

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DNA-protein interactions with cysteines on B-sheets and histidines on A-helix (which binds DNA), stabilized by zinc ion.

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Telomerase

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Adds DNA sequence repeats to the 3' end of DNA to prevent loss of important DNA from chromosome ends (because with each replication, the telomere regions are shortened.)

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TdT

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Terminal deoxynucleotidyl transferase

Used in 5' RACE: adds non-template nucleotides to the 3" end of the DNA

Can make blunt ends sticky

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RACE

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Rapid amplification of cDNA ends

Identify unknown sequence of RNA

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Laz z(alpha)

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The lacZ gene encodes for the alpha subunit of B-galactosidase which serves as the associations/assembly domain

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HDAC

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Histone deacetylase

Enzymes that remove acetyl groups on the lysine amino acids of histones to allow DNA to wrap more tightly

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Paralog

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Two or more different genes in the same species which are so similar in their nucleotide sequences that they are assumed to have originated following the duplication of a single ancestral gene

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E-value

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Expected value

Is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. (eg. BLAST: basic local alignment search tool used for comparing primary sequences of proteins or DNA)

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Southern blot

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A complementary DNA hybridization prode is used to detect specific DNA sequence;
from the blot you can determine which cells incorporated the gene of interest

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Nick translation

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Moving a nick in a phosphodiester bind around

-nick=a break in one strand
-5'->3' exonuclease (DNA pol)
-Tagging tecchnique takes out nick so radioactively tagger dNTPs can be inserted and moves the nick down to the 3' end

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Intercalate

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Inclusion between bases or other molecules (eg Ethidium Bromide in between bases of DNA double helix)

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Tm

Back 37

Temperature at which there is 50% ssDNA and 50% dsDNA