Exam 3 Genetics

PCR purpose

amplifies a specific DNA fragment exponentially using heat-resistant DNA polymerase

PCR temperature steps

95 °C denature separates DNA strands then 55 °C anneal primers then 72 °C extend new strands with Taq polymerase

Taq polymerase origin

enzyme from Thermus aquaticus a hot-spring bacterium that is heat stable and survives repeated heating

PCR enzyme type

DNA polymerase not RNA polymerase

RT-PCR meaning

reverse transcriptase PCR used to convert mRNA into complementary DNA for expression analysis

Reverse transcriptase function

synthesizes complementary DNA from an RNA template

RT-PCR measures

mRNA levels which indicate gene expression rather than total DNA

qPCR purpose

quantitative PCR that uses fluorescence to measure DNA amount in real time

qPCR interpretation

earlier fluorescence curve means more starting DNA or cDNA and higher gene expression

How does Sanger sequencing work?

chain-termination DNA sequencing using fluorescent dideoxynucleotides that stop extension

ddNTP function

lacks a 3 prime hydroxyl group so once added the chain cannot continue growing

Sanger sequencing read length

produces about 500 to 1000 base pair reads of a single DNA fragment

Next-Generation Sequencing definition

massively parallel sequencing of millions of short DNA fragments at once without cloning

NGS read length

produces short reads about 150 base pairs but much faster and cheaper than Sanger

NGS key advantage

no bacterial cloning and can process entire genomes or transcriptomes simultaneously

NGS limitation

short reads make assembling repetitive regions difficult

Paired-end reads purpose

sequences both ends of a DNA fragment to determine distance and orientation for genome assembly

In situ hybridization purpose

uses labeled complementary RNA or DNA probes to show where in a tissue a gene is expressed

RT-PCR vs PCR difference

RT-PCR starts with RNA and measures expression while PCR only amplifies DNA

ddNTP nickname

dead-end nucleotide that terminates DNA synthesis in Sanger sequencing

What is the purpose of the lac operon

to control lactose metabolism genes so they are only made when lactose is present and glucose is low

What do the Z, Y, and A genes of the lac operon code for, and what are their roles?

• Z: β-galactosidase → splits lactose; forms allolactose (inducer).

• Y: Permease → transports lactose into the cell.

• A: Transacetylase → detoxifies sugars (minor role).

What does the I region of the lac operon encode

the lac repressor protein that binds the operator

What is the function of the P promoter region

it is where RNA polymerase binds to start transcription

What is the function of the O operator region

the DNA site where the repressor binds to block RNA polymerase

When does the lac repressor bind to the operator

when lactose is absent

What is positive and negative regulation of gene expression? Give an example using the lac operon.

• Negative regulation: Gene is OFF because a repressor blocks transcription.

  • Example: LacI repressor binds the operator when lactose is absent → no transcription.

  • When lactose/allolactose binds LacI, the repressor is inactivated → transcription turns ON.

• Positive regulation: Gene expression is weak or OFF until an activator helps RNA polymerase bind.

  • Example: CAP–cAMP complex activates the lac operon when glucose is low, boosting transcription.

What happens to the repressor when lactose is present

allolactose binds to it and causes it to unbind from the operator

What role does allolactose play in the lac operon

it acts as an inducer molecule that removes the repressor by binding to it.

What is the effect of high glucose on the lac operon

low cAMP levels make CAP inactive so expression is reduced and Z, Y, and A structural genes are transcribed at a low level

What combination of lactose and glucose gives full lac operon expression

lactose present and glucose absent

What is the difference in lacZ, lacY, and lacA gene expression when glucose is low vs. when glucose is high? (Given Lactose is present)

• Glucose low:

  • cAMP levels rise → CAP–cAMP complex binds the promoter.

  • Lactose (allolactose) inactivates the LacI repressor.

  • RNA polymerase is strongly recruited → Z, Y, and A are highly transcribed.

• Glucose high:

  • cAMP levels drop → CAP–cAMP can’t bind.

  • Even if lactose is present and the repressor is off, RNA polymerase binds weakly.

  • Z, Y, and A are transcribed only at a low (basal) level.

What combination of lactose and glucose keeps the operon off

lactose absent and glucose present

What does an I minus mutation cause

no functional repressor so the operon is constitutively on

What does an I super mutation cause

a repressor that cannot bind allolactose so it stays bound and the operon is always off

What does an O c mutation cause

a mutant operator that cannot bind the repressor so the operon is always on

Which lac operon mutations act in trans and which act in cis?

• I⁻ and Iˢ mutations act in trans (repressor protein can move and affect any operon copy).

• Oᶜ mutations act only in cis (operator sequence affects only genes on the same DNA molecule).

What does CAP stand for and what does it do

catabolite activator protein that binds cAMP to help RNA polymerase bind the promoter

When is the CAP cAMP complex active

when glucose is low and cAMP levels are high

What is constitutive expression

continuous gene expression regardless of environment

What is an inducible system

one that is normally off but turns on when an inducer is present

What two conditions give maximal lac operon expression

lactose present to remove the repressor and glucose absent to activate CAP cAMP