Lab 8: Genotyping Singled Worms

Genotyping (in this experiment)

Using PCR to amplify the region of DNA that CRISPR-Cas9 may have edited, then examining amplicons on a gel to determine whether worms are heterozygous or homozygous for the insert, or do not have the insert at all

Overview of our experiment (genotyping context)

We began with a strain homozygous for unc-119(ox819) with Pmex-5::Cas9.

We injected a plasmid with sgRNA + repair template and a plasmid with coel::gfp.

F1 with co-injection marker were kept.

Students singled progeny (F2/F3), then genotyped the singled worm to find worms homozygous for the desired insertion

Why genotype the singled worm

If we know the genotype of the parent that was placed on the plate, we can predict the possible genotypes of the progeny that remain on the plate

What a successful insertion looks like on a gel

Worms with an insertion of DNA in the amplified region will have a band that runs higher on the gel (larger molecular weight) compared to wild-type.

A worm homozygous for an insertion shows a single higher band

PCR (Polymerase Chain Reaction)

A method by which a defined region of DNA is synthesized in large quantities (DNA replication in a test-tube); powerful enough to amplify minute quantities of DNA, revolutionizing molecular biology, genetic diagnosis, forensics, and basic research

Principal requirements for PCR reaction

1. DNA template (sequence to be amplified),

2. short (18-22 bp) oligonucleotide primers specific for the region of interest,

3. DNA polymerase,

4. four deoxynucleoside triphosphates (dNTPs),

5. appropriate reaction conditions (buffering and Mg cations) for the DNA polymerase

Taq polymerase

A thermally stable DNA polymerase isolated from Thermus aquaticus; used in PCR because it can withstand the high temperatures of denaturation steps

Thermocycler

A programmable temperature block (PCR machine) that accurately heats and cools samples many times to perform the denaturation, annealing, and extension steps of PCR

DNA polymerase directionality

DNA polymerase adds nucleotides to the 3' end of DNA (not the 5' end)

Initial Denaturation Phase

94-98°C for 10 to 60 seconds;

DNA is heated and separates (denatures) into single strands.

A short denaturation step precedes the cycles to ensure template DNA is denatured.

Higher GC content requires longer denaturation time, but long times risk denaturing the polymerase

Denaturation step (cycling phase)

94-98°C for 10 to 60 seconds; the temperature is raised to separate double-stranded DNA into single strands.

Time is kept as short as possible to prevent damaging the polymerase

Annealing step (cycling phase)

55°C - 65°C; the temperature is lowered to allow primers to bind to their complementary sequences on the single-stranded DNA template.

Classically, the annealing temperature should be about 5°C below the Tm of the primers

Extension step (cycling phase)

72°C; the elongation temperature is raised to approximately the optimal temperature for DNA polymerase to synthesize DNA.

General rule: 60 seconds of extension time for each 1kb of sequence. Minimum 45 seconds even for small pieces of DNA

Final Extension Phase

72°C for approximately 5 minutes; allows any incomplete strands to complete their elongation after all cycles are completed.

Some Taqs add an A at the final base addition, which can be useful for cloning

Number of PCR cycles

25-35 cycles are necessary to synthesize enough DNA for analysis. Including more cycles gives higher yield but also risks amplifying byproducts

Exponential amplification in PCR

PCR amplifies DNA exponentially, doubling the template number each cycle. After n cycles, there are 2^n copies of the target sequence

Primers (PCR)

Short (18-22 bp) oligonucleotides that flank the target region; they are complementary to short stretches of DNA sequence on either side of the CRISPR insertion. Primers anneal to DNA at regions flanking the sequence to be amplified

Primer complementary requirement

The primer sequence must be complementary to the DNA sequence where it needs to bind

Primer GC content requirement

Primers must have enough GC content to make stable pairing; 40% - 60% GC content is considered acceptable

Primer melting temperature requirement

The two primers must have similar melting temperature (Tm), differing by no more than 5°C

Primer secondary structures

Primers should not form secondary structures (hairpins) within themselves, and the two primers should not complement/bind one another (creates primer-dimer).

It is especially important that the 3' end of a primer not contain hairpins/self-complementarity

Primer length

16-30 nt long; 18-22 nt is the usual length

Primer 3' GC clamp advantage

It is advantageous for the last 5 nucleotides at the 3' end of a primer to contain 3 G or C to better "clamp" the primer onto the template.

If the double strand begins to fray, it occurs at the ends, so the 3 bonds in a G-C pair are helpful

Primer specificity requirement

Primers should be specific to the region of interest so other regions will not be amplified.

Matches to undesired sequences at the 3' end of a primer are most problematic

Why clamping the 3' end of a primer is more important than clamping the 5' end

DNA polymerase adds nucleotides to the 3' end; if the 3' end is not firmly bound, the polymerase may not extend efficiently or may extend from mismatched sites, leading to non-specific amplification

Primer orientation requirement

Both primers, when bound, must point 'inward' in the 5' to 3' direction towards the region to be copied because DNA polymerase can only synthesize DNA in the 5' to 3' direction

Three methods to lyse C. elegans

Detergents (break apart lipids, creating holes in cell membranes),

enzymatic Proteinase K (cleaves all proteins, destroying nucleases and exposing DNA),

and freeze-thaw cycles (cracks the proteoglycan-rich cuticle)

Why multiple lysis methods are needed for C. elegans

C. elegans is surrounded by a proteoglycan-rich cuticle that is difficult to break; freeze-thaw cracks the cuticle, detergents break cell membranes, and Proteinase K digests proteins surrounding DNA in chromatin and destroys endogenous nucleases

Proteinase K functions in worm lysis

A broadly non-specific protease that cleaves all proteins; serves two functions:

(1) breaks down proteins surrounding DNA in chromatin so PCR reactants have access to DNA, and

(2) destroys endogenous nuclease activity (essential for RNA work)

Worm lysis buffer components (activated)

Lysis buffer (contains detergent) plus Proteinase K at a 25:1 ratio (Lysis Buffer : Proteinase K)

Freeze-cracking method

Worms in lysis buffer are placed in an ethanol-dry ice bath for about 2 minutes; the extreme cold freezes and cracks the cuticle, then samples are transferred to a thermocycler at 60°C for Proteinase K activity

Thermocycler worm lysis protocol conditions

60°C for 1 hour (optimal ProK activity), then 95°C for 15 minutes (heat inactivates Proteinase K and denatures DNA), then 4°C indefinitely

Why 95°C for 15 minutes after ProK treatment

Heat inactivates Proteinase K (so it doesn't degrade the Taq polymerase later) and denatures the DNA to make it single-stranded and accessible for PCR priming

Phusion Taq polymerase

A modified Taq polymerase with a custom buffer that requires higher annealing temperatures because melt temperatures are higher in this buffer

PCR master mix

Instead of mixing separate reactions in separate tubes, a master mix combines all common reagents (water, buffer, dNTPs, primers) which is then dispensed into individual tubes; saves time and reagents and avoids pipetting very small volumes. Taq and lysate are added separately

Primer design programs

Online tools (available on primer-selling sites and NCBI) where you enter target nucleotide sequence and the program returns candidate primers with melt temperatures indicated; Tm is primarily determined by GC content and length

Variables determining annealing temperature and time

Length of primer sequences, GC content of the primers, primer concentration, and buffering conditions

Extension time rule of thumb

60 seconds of extension time for each 1kb of sequence being amplified;

minimum extension time recommended is 45 seconds even for small pieces of DNA

MgCl2 role in PCR

Magnesium ions are an essential cofactor for DNA polymerase. Too low concentration results in weak amplification or PCR failure; too much Mg++ can cause non-specific nucleotide interactions leading to non-specific amplification

Standard starting MgCl2 concentration for PCR

1.5 mM is the standard starting concentration for PCR reactions

Phusion Taq primer concentration recommendation

0.5 uM, which is higher than for most other Taqs

Definition of one unit of Taq enzyme activity

Varies by manufacturer but typically: the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 74°C (for the polymerase used in this lab)

How to pipette viscous solutions (like lysis buffer with detergent)

Pipette slowly and carefully; eject more slowly to minimize bubbles. The tween detergent makes the solution "soapy"

Why use human DNA or E. coli as a control

To serve as a negative control (should not amplify with worm-specific primers) to detect contamination or non-specific amplification; can also perform this control in silico by checking primer specificity using BLAST against other genomes

What to do if the singled worm cannot be found

Choose one worm (likely an F1 progeny) to transfer into lysis buffer, and note that in the lab notebook. Record that the worm may not be the original parent

What to record about each lysate tube

How many worms are in the tube (check under dissecting microscope), features of the transferred worm (phenotype: Unc or wild-type), whether confident it was the original parent or could be an F1, and which plate each tube corresponds to

Why save leftover lysates

For potential repeat PCR reactions if the first attempt fails or needs confirmation

Phusion Taq PCR cycling conditions

98°C for 2.5 min initial denaturation; then 26-30 cycles of: 98°C for 8 sec (denaturation), 64°C for 20-30 sec (annealing), 72°C for 120 sec (extension for genomic DNA template); then 72°C for 5-10 min final extension; hold at 4°C

Why higher MgCl2 concentration might be tried

Higher MgCl2 can increase PCR yield, but may also create non-specific bands. Some students may try 2.0 or 2.5 mM if standard 1.5 mM gives weak amplification

How to label tubes for PCR

Use ethanol-resistant marker for tubes that will go into ethanol-dry ice bath; normal Sharpie marks may come off. Regular Sharpie can be used after removing ethanol residue

What to do if multiple worms are in a lysis tube

Note that information in the lab notebook; having multiple worms means the genotype result may represent a mixture of individuals

What to check before placing tubes in dry ice bath

Ensure caps are tightly sealed (listen for the "click" sound); TAs should check because about a third of students do not seal lids properly

Melting temperature (Tm) definition

The temperature at which 50% of the DNA molecules (or primer-template duplexes) are denatured (single-stranded)

Main determinants of primer melt temperature

GC content and length of the primer sequence. Higher GC content increases Tm because G-C pairs have 3 hydrogen bonds versus 2 for A-T pairs

How often a 20-mer oligonucleotide matches a target sequence by chance

Assuming equal probability of A, T, G, C (1/4 each), the chance is (1/4)^20 = 1 in 1,099,511,627,776 (approximately 1.1 trillion). For a 1-mer: 1/4; 2-mer: 1/16; etc.

Why a primer might produce two robust bands (non-specific amplification)

Not likely due to random chance (extremely low probability).

More systematic reasons include: the primer has partial complementarity to another genomic region (especially at the 3' end), or the second band represents a different isoform or paralogous gene with high sequence similarity

What to do if the singled worm was heterozygous and an F1 was genotyped instead

If the original singled worm was heterozygous for the GFP insert, the ratio of F1 worms that contain the GFP insert to those that do not is 1:1 (50% contain the insert, 50% do not) because the parent is m/+ and self-fertilization produces 1/2 m/+ and 1/4 +/+ and 1/4 m/m, but if picking an individual F1 worm, probability of having the insert is 1/2 (the heterozygotes)

How to determine if a plate has worms homozygous for the insertion

Run PCR products on a gel; a worm homozygous for an insertion will show a single band that runs higher (larger) than the wild-type band. A heterozygous worm will show two bands (both wild-type and insertion sizes)

Why we use both detergents and Proteinase K

Detergents break lipid membranes; Proteinase K digests proteins including histones that package DNA (releasing DNA from chromatin) and destroys nucleases that would degrade DNA. Both are needed for efficient DNA release and preservation

Why we use freeze-thaw cycles specifically for C. elegans

The proteoglycan-rich cuticle of C. elegans is tough and resistant to detergents and enzymes alone; freeze-thaw physically cracks the cuticle, allowing the detergents and Proteinase K access to cells inside the worm

PCR annealing temperature for Phusion vs regular Taq

Phusion Taq requires higher annealing temperatures (e.g., 64°C) because the Phusion buffer increases melt temperatures; regular Taq typically uses 50-60°C annealing temperatures

Effect of higher primer concentration on annealing temperature

Higher primer concentrations allow lower annealing temperatures because more primers are available to bind even at less-stringent conditions. Conversely, lower primer concentrations may require higher annealing temperatures to maintain specificity

What the gray and blue boxes in the repair template figure represent

The gray box (left) and blue box (right) represent the homology arms (60 nucleotides each) that match the target DNA sequence adjacent to the DSB; these flank the GFP and unc-119 insertion

Will successfully altered worms have an Unc-32 mutant phenotype?

No (or not necessarily). If the GFP tag is inserted at the C-terminus with a linker sequence and does not disrupt protein function, the worms will not have an Unc-32 phenotype. The goal is to add the tag without disrupting UNC-32 function

Why injected worms are unc-119(ox819) but successfully edited worms are not Unc

Injected worms are unc-119(ox819) (mutant) so they are Unc. The repair template contains wild-type unc-119 within a gfp intron. Successful CRISPR editing inserts both gfp and wild-type unc-119, which rescues the Unc-119 phenotype, so edited worms are non-Unc

What proportion of progeny will be homozygous for the gfp::unc-119 insertion if a heterozygous worm reproduces

If a worm is heterozygous for the gfp::unc-119 insertion (i.e., has one edited and one wild-type chromosome), self-fertilization produces 1/4 homozygous for the insertion, 1/2 heterozygous, and 1/4 wild-type. So 25% of progeny will be homozygous for the insertion

NEB Tm Calculator

An online tool (tmcalculator.neb.com) where you can enter primer sequences to calculate recommended annealing temperatures for different polymerase buffers (Q5, Phusion, regular Taq) at specific primer concentrations

Definition of a genetic mosaic (PCR context)

An individual composed of cells with different genotypes; for the co-injection marker plasmid (unstable inheritance), some cells inherit the plasmid and express GFP while others do not, creating a mosaic pattern even in F1 worms

Why we use both forward and reverse primers

Both primers are needed because DNA polymerase can only synthesize in the 5' to 3' direction; the forward primer binds to the template strand and extends toward the reverse primer, while the reverse primer binds to the coding strand and extends toward the forward primer, amplifying the region between them

What the "wobble" position in a codon allows

Allows silent mutations to be made in the PAM or protospacer sequence without changing the amino acid sequence; the third nucleotide of a codon can often be changed without altering the encoded amino acid due to redundancy in the genetic code

How to calculate primer concentration in PCR reaction

Use C1V1 = C2V2. For example: if adding 1.0 µL of 10 µM primer to a 25 µL final reaction: (10 µM)(1 µL) = C2(25 µL) → C2 = 0.4 µM (400 nM)