Exam 2 - PCR, Plasmids, Transcription, and Translation

What is PCR?

polymerase chain reaction

What are the steps of PCR

denaturation, annealing, extension, repeat

What is denaturing in PCR

The use of heat to separation the DNA strand into two different pieces. This

What is annealing in PCR

Bonding primers onto the target locus

What is extension in PCR

the addition of free nucleotides that are complementary to the template onto the 3' end of the primer

What happens every cycle of PCR

DNA doubles

What is gel electrophoresis?

the separation of charges molecules using an electric field

What is PCR used for?

amplify DNA

What is gel electrophoresis used for?

Organize DNA fragments by size

How can fluorescence be used in PCR

Determine how many relative copies exist

What is Illuminati sequencing

Finding the exact base sequence of a PCR sample

How do you edit a plasmid

- Cut open backbone at MCS using restriction enzymes

- ligate target dna

- 1 add complimentary primers around area of interest

- PCR to duplicate

What is a multiple cloning site

Area with repeated sites for restriction enzymes

What is used to cut plasmids

restriction enzymes

What is a selectable marker?

Genes used to determine if cells have successfully taken up DNA

lac operon

LAC binds to promoter and represses until in presence of lactose, changes color

What is a motif

A highly conserves stretch of DNA that binds specific proteins

What is a promoter

Section before coding sequence that tells polymerase where to bind

What is the difference between promoters in eukaryotes vs prokaryotes

Eukaryotes need 1 per gene while prokaryotes can have 1 for mutable genes

What is a constitutive promoter

A promoter that is always active

What is a negative inducible promoter

Turns off when exposed to signal

What is a positive inducuble promoter

Activates when exposed to signal

What is a terminator

signals the end of transcription

Why is an enhancer

Enhances promoter, loops and amplifies what's inside

What are transcription factors

Protein that bind to dna and amplify or negate nearby sequences

What is heterochromatin?

highly condensed and dark protein and DNA

What is Euchromatin

less condensed, transcriptionally active DNA

What strand does RNA polymerase read?

antisense strand / template / 3 to 5

What is pre initiation complex (PIC)

Poly II and friends

What is splicing

Removing introns from RNA and sealing exons together

What is the purpose of the 5' cap

Transport to ribosome, protection from exonuclease

What is the purpose of Poly A tail

Protection from exonuclease

What does poly A binding protein do

Attaches to poly A tail and stabilizes

What is the difference between mRNA and ncRNA

nc is non coding m is coding

How does PCR of mRNA work

Half on coding half on poly A tail

What is co-transcriptional editing

RNA editing that happens while transcription occurs

What is post transcriptional editing

RNA editing that happens after transcription

Where do elongation factors attach

Poly II

Wha are two elongation factors

NELF and P-TEFb

What does NELF do

Block Poly II

What does P-TEDb do

Remove NELF

What does TFIIS do?

Proofread rna, if mistake reverse and retry

What is alternative splicing?

Variations to splicing that result in different proteins with similar functions

What are antisense oliglonucleotides

Complimentary pairs that stop RNA from being translated

What is the difference between siRNA / shRNA and microRNA

Micro RNA is natural si/sh are lab grown

What does mod RNA do

Increase stability of RNA ex: COVID

What are the two controls how much of a protein gets made

How much of rna is made, how much protein each rna makes

What are four things that affect how much rna is made

Promoters, enhancers, elongation factors, transcription factors

What are two things that effect how much protein is made from an rna

ASOs, exonucleases

What strand does the ribosome read

Template / 3-5

Where are ribosomes made

nucleolus

What is the Shine-Dalgarno sequence?

Ribosome binding site on prokaryotes

What is the kodak sequence?

Ribosome bonding site in eukaryotes a

Cell

What are the three sites of a ribosome

E, P, A

What is a poly some

A single mRNA connected to multiple ribosomes

What are two post translational modifications

Kinase and phosphotates

What do kinases do

add a phosphate group

What do phosphatases do?

remove phosphate group

What is ubiquitination?

Targeting of a protein for degradation by proteasome

What is a protesome?

Destroys damaged or unwanted cellular material

What is the active site of an enzyme?

The site that binds to the thing the enzyme works on

What is the allosteric site of an enzyme

Controls shape of protein

What does a chaperone protein do

Allows proteins to fold without outside influence

How do you perform a western blot

Denature into linear proteins and coat with charged substance, run charge through to sort by size, use antibodies to observe against ladder

What are monoclonal antibodies?

A single antibody that binds to a single epitope

What are polyclonal antibodies

A mix of antibodies that bind to multiple epitopes

What is an epitope

Specific region of a protein