Genetics Lab Final Review

Polymerase Chain Reaction (PCR)

A laboratory method used to amplify a specific segment of DNA, producing millions of copies

Purpose of PCR

To replicate a specific DNA region in vitro for analysis, identification, or genetic testing

Semi-conservative replication

Each new DNA strand is built using one of the original strands as a template

Thermal Cycler

Machine that automatically cycles through temperatures for PCR denaturation, annealing, and extension

Denaturation Step

DNA strands separate at 94°C by breaking hydrogen bonds between base pairs

Annealing Step

Primers attach to their complementary DNA sequences at ~55°C

Extension Step

Taq polymerase adds nucleotides to synthesize new DNA strands at 72°C

Pre-Denaturation Step 

Ensures all DNA is fully separated before cycling begins

Final Extension Step

Allows any incomplete strands to finish elongating

Cycle Count

30–40 cycles, producing over a billion copies of the target sequence

Template DNA

The DNA sample containing the target region to be amplified

Primers

Short single-stranded DNA pieces that mark the start and end of the region to be copied

dNTPs (Deoxynucleotide Triphosphates)

The building blocks (A, T, G, C) used by polymerase to create new DNA strands

Taq Polymerase

Heat-stable enzyme from Thermus aquaticus that synthesizes DNA at high temperatures

Mg2+ Ions

Cofactor that activates DNA polymerase and stabilizes primer-template binding

Buffer

Maintains stable pH and ionic strength for enzyme activity

Chelating Agent

Binds metal ions (like Mg²⁺) to inhibit DNase enzymes and protect DNA during extraction

Saline Mouth Rinse

Collects epithelial cheek cells containing genomic DNA

Centrifugation

Spins cells into a pellet at the bottom of the tub

Gel Matrix + Protease

Breaks open cells and digests proteins to release DNA

56*C Incubation

Helps lyse cells and activate protease enzymes

100*C Incubation

Denatures proteins and enzymes that could degrade DNA

Supernatant

Liquid containing purified DNA after centrifugation

PV92 Locus

A specific site on chromosome 16 often used in genetics labs to detect the presence of an Alu insertion

Alu Sequence 

A 300 bp short interspersed element (SINE) repeated ~500,000 times in the human genome

Dimorphic Trait

A genetic locus with two possible forms (with or without the Alu insertion)

Homozygous (+/+)

Both chromosomes have the Alu insertion → band at 941 bp

Homozygous (-/-)

No Alu insertions → band at 641 bp

Heterozygous (+/-)

One chromosome has the Alu insertion → two bands (941 bp & 641 bp)

Purpose of Electrophoresis

To separate DNA fragments by size using an electric current through an agarose gel.

Agarose Gel

A porous matrix that slows larger DNA fragments more than smaller ones

SYBR Safe DNA Stain

A fluorescent dye that binds to DNA and allows visualization under UV light

Loading Dye

Adds color and density to samples so they sink into the wells and migration can be tracked

Electric Field Direction

DNA migrates toward the positive (anode) because it’s negatively charged

Base Pairs (bp)

Units of DNA fragment length; the distance between nucleotide pair

Band

A visible DNA fragment on a gel corresponding to a specific size

Genotype

The combination of alleles present at a specific locus (e.g., +/–)

Phenotype

The physical or observable outcome (in this lab, the presence or absence of a band pattern)

Non-coding Regions (Introns)

DNA sequences that do not code for proteins; often contain insertions like Alu elements

Bioinformatics

The use of computational tools to analyze and interpret biological data like DNA sequences.

Allele Frequency

The proportion of each allele variant in a population.

FST (Genetic Distance)

A statistical measure of genetic variation between populations;
0 = identical populations, 1 = completely different.

Genotype Frequency 

The proportion of each genotype (++, +–, ––) in a population.

Neighbor-Joining Method

A computational approach used to create phylogenetic trees based on genetic distance data.

Why was Taq polymerase a breakthrough for PCR?

It is heat-stable and doesn’t denature during high-temperature steps.

What would happen if the annealing temperature is too high?

Primers won’t bind efficiently → low or no amplification.

What would happen if the annealing temperature is too low?

Primers may bind non-specifically → incorrect products.

Why are introns good regions to target for variation studies?

They’re non-coding and tolerate mutations or insertions like Alu elements.

How can PCR data be used to compare human populations?

By measuring allele and genotype frequencies and computing genetic distances (FST)

What makes the PV92 locus useful for teaching?

It’s harmless, dimorphic, and easily amplified for visible genetic variation.

Why can’t one locus identify a person in forensics?

It’s not unique enough; need multiple loci for reliable identification.

What does a small FST value mean?

Populations are genetically similar or recently diverged

What does a large FST value mean?

Populations are genetically distinct or long separated.

How does PCR connect molecular and population genetics?

It amplifies DNA for individual genotyping, allowing analysis of population-level allele differences.

Mutation

A change in the DNA sequence that can occur naturally or be induced in the lab.

Purpose of Mutation Studies

To understand gene and protein function by intentionally altering DNA sequences.

Nonsense Mutation

Changes an amino acid codon into a stop codon, causing premature termination of translation.

Missense Mutation

Changes one amino acid into another within the protein.

Silent Mutation

Does not alter the amino acid sequence due to redundancy in the genetic code.

Frameshift Mutation

Insertion or deletion of nucleotides not in multiples of three, shifting the reading frame.

Site-Directed Mutagenesis

A lab technique used to introduce specific mutations into a DNA sequence at a chosen site.

Quick-change Technique

A method of site-directed mutagenesis that uses primers to introduce mutations without ligation.

Quick Change Advantages 

• Works on any dsDNA plasmid

• No ligation required

• Fast and efficient (>80% transformation efficiency)

Key Enzymes in Quick Change

• PfuTurbo DNA polymerase: synthesizes new DNA with high fidelity

• DpnI restriction enzyme: digests methylated (parental) DNA only

Dpnl Recognition Sequence

GATC — only cuts when at least one strand is methylated (5’-Gm6ATC-3’).

Why Dpnl is Used

To remove the original, methylated plasmid DNA and leave only the newly synthesized mutant plasmid.

Why PfuTurbo is Used Instead of Taq

PfuTurbo has proofreading ability and higher fidelity (6× more accurate), ideal for amplifying plasmids.

Primers in QuickChange

Two complementary primers designed with the desired mutation near the center.

Purpose of Cooling tubes

Stops the reaction and stabilizes the DNA. Without cooling, enzymes might continue reacting, degrading the DNA.

pUC19 Plasmid

A common plasmid containing the lacZ gene used for blue-white screening.

Purpose of Site-Directed Mutagenesis I

To insert one nucleotide into the lacZ gene to create a frameshift mutation that disrupts β-galactosidase activity.

Purpose of Site-Directed Mutagenesis II

To test the effects of the frameshift mutation in lacZ by transforming E. coli and observing colony color.

B-galactosidase Function

Enzyme that breaks down X-gal into a blue pigment when functional.

Blue-White Screening 

Technique to identify recombinant or mutant plasmids using colony color on X-gal plates.

Functional B-galactosidase (Blue Colonies)

indicates the lacZ gene is intact and functional.

Nonfunctional B-galactosidase (White Colonies)

Indicates the lacZ gene is disrupted (mutation present)

α and Ω Subunits

• α (from pUC19 plasmid)

• Ω (from E. coli chromosome)
  They must combine for functional β-galactosidase activity.

X-gal

A substrate that produces a blue pigment when cleaved by β-galactosidase.

Reaction w/ DpnI (Experimental)

Expected result: White colonies — parental DNA digested, mutant plasmid remains.

Reaction w/o DpnI (Experimental)

Expected result: Mix of blue and white colonies — both parental and mutant plasmids present.

Control w/ DpnI

Expected result: No colonies or very few — parental plasmid digested, no mutation introduced

Control w/o DpnI

Expected result: Blue colonies — parental plasmid intact and functional.

Purpose of Ice Incubation (Cooling on Ice)

Helps DNA bind to the cell membrane before heat shock; skipping it reduces transformation efficiency.

Purpose of Heat Shock (42°C, 45 sec)

Creates temporary pores in bacterial membrane to allow plasmid entry.

LB Broth Incubation

Allows bacteria to recover and express antibiotic resistance genes before plating.

Ampicillin in Agar Plates

Selects for cells containing the plasmid (pUC19 has an ampicillin resistance gene).

BamH1 and EcoR1 jobs

cut the multicloning site of the plasmid; done to create an open DNA structure so other DNA can be inserted through a process called ligation

ligation

process in which DNA is inserted into an open part of a DNA structure

buffers

used to establish constant conditions in the reaction

mutation at codon 12

expression of a faulty protein where GGC (glycine) í GTC(valine) leading to amino acid glycine to be replaced by valine

point mutation

single nucleotide base substitution; alters protein structure and function

sticky ends

overhanging ssDNA on ends

annealing

process of creating dsDNA from complementary ssDNA; or the hydrogen bonding of 2 single DNA strands to form a double strand

Why do we heat ssDNA before cooling to anneal them?

to denature any hydrogen bonds formed from folding within itself to assure we can let the DNA anneal properly with a second strand of DNA

plasmid

circular piece of DNA

Poly-linker / multicloning site

part of plasmid DNA that has multiple restriction endonuclease sites that will allow for cutting and the inserting of foreign DNA into the plasmid

restriction endonuclease digestion

the cutting process of DNA

antibiotic resistance

a means to easily select bacteria that has taken up a DNA plasmid making it resistant to the antibiotic

blunt ends

DNA that is cut with no sticky ends produced

competent bacteria

bacteria that has been chemically treated to make them leaky and thus easier for DNA to enter through the outer membrane