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Effect of single nucleotide deletion or insertion on primer annealing

Effect of single nucleotide deletion or insertion on primer annealing


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How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ?

Imagine a primer like this:
GCGTCATAAAGGGGACGTG (primer)
and the corresponding part of template DNA has one G missing, so it looks like this:
GCGTCATAAAGGGACGTG (template).

The possible pairing could be
GCGTCATAAAGGGGACGTG primer
GCGTCATAAA_GGGACGTG template
or
GCGTCATAAAGGGGACGTG primer
GCGTCATAAAGGG_ACGTG template
or anything in between.

Is it possible that amplification with such primer would be completely disrupted in normal real time PCR at 60°C annealing? Could this completely disrupt the amplification?

If the mismatch was substitution-like, I would be pretty confident, the primer would be still functional and amplification would occur. In the extreme case, it would be at least residual amplification at late Ct. There are a lot of data, how substitution mismatches affect primers and I also have a lot of personal experience with that.

Unfortunately, the deletion mismatches are less studied plus googleproof. The only indication I have found is this work:
Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, et al. DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem. 2001;47:635-44.
In that work, single nucleotide deletion had similar or lower affect on melting temperature than substitution mismatch. But it was about longer oligos. Examples:
Effect of deletion:
133 bp fragment, 67 % GC, deletion SNP at position 43, delta Tm (homo-hetero duplex) = 1.2°C
Effects of substitutions:
152 bp fragment, 43 % GC, substitution T to C at position 68, delta Tm (homo-hetero duplex) = 0.9°C
100 bp fragment, 41 % GC, substitution T to C at position 42, delta Tm (homo-hetero duplex) = 1.4°C
163 bp fragment, 60 % GC, substitution C to T at position 86, delta Tm (homo-hetero duplex) = 2.2°C
110 bp fragment, 59 % GC, substitution G to A at position 66, delta Tm (homo-hetero duplex) = 3.8°C

Questions:
1. Can you recommend me literature about how deletion mismatches inside (not at the very end of !!!) primers affect annealing and PCR ?
2. Would You guess the primer in my example would be still functional , at least partly, or would You expect no amplification at all ?


EDIT after your inputs:
This online application " mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting " thinks, deletion mismatches are more destabilising than substitutions, at least for short primers. For my own example, it calculated delta Tm (homo-hetero duplex) = 12.9°C . If I try substitution mismatches instead, delta Tm (homo-hetero duplex) is in interval 3,8°C to 5,7°C.

New question
If you have experience as similar as possible to my case, which is 19 nt long primer with single nucleotide deletion in the comlementary template at position cca 6 - 9 from 3' primer end, annealing temperature used at 60°C, please let me know if You achieved amplification or not. Please, give me a respective reference, if you have it, so I will quote it in my review :-) .

Also, I am still interested in general info, as long as the topic is narrow enough to be about single nucleotide deletions or insertions in primers. (Not substitution mismatches).


We used this kind of primers to generate out of frame mutations or to add additional bases. In my experience your PCR will work (probably a a lower efficiency) and you will get a product with an additional base. We used primers with bigger differences in PCR based site directed mutagenesis of plasmids, there up to 10 bases didn't match but the primers where also longer. For single nucleotide mismatches (either + or - one base) we used primers around this size.

Regarding the literature, this publications might be useful:

Especially the first publication contains a lot of other interestings references.


Adding another reference. This group looks at the effect of different mismatches (e.g. A>T vs A>G) and also looks at positional effects.

Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797725/


An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.

Results

We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.

Conclusion

Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™) but increased the overall success rates.


Abstract

Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1–5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3’ end are partially corrected and extended. Indels located 7–9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.


An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.

Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.

Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.


Results

Assay development

We evaluated two thermostable polymerases designed for dideoxynucleotide incorporation to assess their capacity for allelic discrimination: Thermo Sequenase (Thermus aquaticus DNA polymerase F667Y) and Therminator (Thermococcus 9°N-7 DNA polymerase A485L). Figure 2 presents the specific terminator incorporation by these enzymes at a series of G/A SNPs, as a function of terminator concentration. PCR and cleanup of genomic DNA templates were as described under methods, with extension in the presence of supplied buffers and 10 mM ddNTP terminators. We assessed specific incorporation as the difference between the incorporation signals of the complementary and noncomplementary (incorrect) terminators. At high enzyme concentrations, nonspecific incorporation can be observed. With these initial tests, greatest specific incorporation was observed at extension enzyme concentrations of 0.004 units/μl for Therminator and, 0.02 units/μl for Thermo Sequenase.

Extension polymerase concentration curves. (A) Specific incorporation as a function of Therminator concentration. (B) Specific incorporation as a function of Thermo Sequenase concentration.

Extension polymerase concentration curves. (A) Specific incorporation as a function of Therminator concentration. (B) Specific incorporation as a function of Thermo Sequenase concentration.

Both Thermo Sequenase and Therminator showed expected assay performance variation across different SNPs (e.g. Supplementary Fig. S2 ). As an aid to assay optimization, we devised a synthetic target system to provide control over template and variant site context. These targets encompassed flanking PCR priming site sequences as well as sufficient sequence surrounding a variant position for subsequent hybridization of an extension primer, facilitating terminator incorporation at the variant position. Examples of synthetic target template assay performance are shown in Fig. 3.

Example assay of a synthetic target template (SynFP_C and SynFP_T) as an engineered SNP using Therminator (A) or Thermo Sequenase (B).

Example assay of a synthetic target template (SynFP_C and SynFP_T) as an engineered SNP using Therminator (A) or Thermo Sequenase (B).

We investigated the impact of extension buffer composition upon terminator incorporation by Therminator. We employed synthetic targets SynFP_C and SynFP_T (incorporating ddG-R110 and ddA-TAMRA) and an initial buffer of 2 mM, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, and 20 mM Tris-HCl pH 8.8. Our approach was to test a range of concentrations of one component, each in the presence of a range of concentrations of a second component, while holding other variables constant. We selected the optimum for each of the two tested components, adopting the new condition as a change to the initial buffer. We followed this approach until optima for each variable had been selected. The optimized reaction was 0.5 μM extension primer and 1× buffer A: 2 mM MgSO4, 5 mM (NH4)2SO4, and 0.1% Triton X-100, and 20 mM Tris-HCl pH 9.3. Thermo Sequenase also performed well in these conditions, though we later analogously optimized buffer B for it (described further below).

We next evaluated the efficiency of ddU-TAMRA, ddG-R110, ddC-R110, and ddA-TAMRA terminator incorporation by both Therminator and by Thermo Sequenase in buffer A using four corresponding synthetic targets ( Fig. 4). Only Thermo Sequenase incorporated all four of these terminators efficiently, and so was chosen for all subsequent experiments. For Thermo Sequenase, optimal 1× terminator concentrations of these four terminators were 35 nM ddA-TAMRA, 4 nM ddC-R110, 4 nM ddG-R110, and 10 nM ddU-TAMRA ( Fig. 5). We further optimized an extension buffer specifically for Thermo Sequenase using synthetic targets and the general approach outlined above ( Fig. 6). The resulting 1× buffer B contained: 6 mM MgSO4, 5 mM (NH4)2SO4, 0.05% Triton X-100, 20 mM Tris-HCl pH 8.9, and the addition of 5% glycerol. The Thermo Sequenase concentration at which terminator incorporation was most specific was 0.0175 units/μl. An additional two terminators were also evaluated, choosing optimal concentrations of 4 nM ddU-R110 and 20 nM ddC-TAMRA for Thermo Sequenase ( Fig. 7). Even with efficient and specific terminator incorporation, the accumulation of labeled extension primer is a function of the number of linear thermal cycles. The sum of the specific signals of both possible extension products (FP sum) of an assayed SNP plateaued at roughly 26 extension cycles (illustrated in Supplementary Fig. S3 ).

Specificity of fluorescently labeled ddNTP terminator incorporation by Therminator and Thermo Sequenase, evaluated using synthetic targets SynFP_A, SynFP_C, SynFP_G, and SynFP_T.

Specificity of fluorescently labeled ddNTP terminator incorporation by Therminator and Thermo Sequenase, evaluated using synthetic targets SynFP_A, SynFP_C, SynFP_G, and SynFP_T.

Specificity of terminator incorporation by Thermo Sequenase as a function of ddNTP concentration, evaluated using synthetic targets SynFPz_A, SynFPz_C, SynFPz_G, and SynFPz_T.

Specificity of terminator incorporation by Thermo Sequenase as a function of ddNTP concentration, evaluated using synthetic targets SynFPz_A, SynFPz_C, SynFPz_G, and SynFPz_T.

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Specificity of terminator incorporation by Thermo Sequenase as a function of buffer components and enzyme concentration. Each panel presents an evaluated reaction component curve for the selection of an optimum.

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Specificity of terminator incorporation by Thermo Sequenase as a function of buffer components and enzyme concentration. Each panel presents an evaluated reaction component curve for the selection of an optimum.

Specificity of ddC-TAMRA and ddU-R110 incorporation by Thermo Sequenase as a function of ddNTP concentration, evaluated using synthetic targets SynFPz_A, SynFPz_C, SynFPz_G, and SynFPz_T.

Specificity of ddC-TAMRA and ddU-R110 incorporation by Thermo Sequenase as a function of ddNTP concentration, evaluated using synthetic targets SynFPz_A, SynFPz_C, SynFPz_G, and SynFPz_T.

Incorrect terminator incorporation becomes problematic with the use of decreasing concentration of either Exo I or CIAP for post-PCR cleanup ( Fig. 8). Residual PCR primers and residual dNTPs can allow incorporation of a labeled terminator at a position other than the intended, interrogated variant site. The optimal concentration of each enzyme for specific terminator incorporation was 0.95 units per reaction, with no difference between heat inactivation at 80°C for 15 min versus 95°C for 30 min.

Effect of CIAP and Exo I concentration on terminator incorporation specificity. Incorporation of specific and nonspecific fluorescently labeled ddNTP terminators are illustrated for synthetic template targets SynFP_G (A) and for SynFP_A (B).

Effect of CIAP and Exo I concentration on terminator incorporation specificity. Incorporation of specific and nonspecific fluorescently labeled ddNTP terminators are illustrated for synthetic template targets SynFP_G (A) and for SynFP_A (B).

Assay performance with production genotyping

We applied the iteratively optimized SNuPE assay to a set of 98 SNPs and indels, designing assays for each to genotype 2202 DNA samples. We selected PCR conditions for each using the approach described under Methods 87 used AmpliTaq Gold/no betaine, 9 used AmpliTaq Gold/betaine, and 2 used Titanium Taq/betaine. For each desired variant assay, we then amplified synthetic targets designed to represent AA, AB (mixed), and BB genotypes for comparison of forward and reverse extension assay performance. The version with greatest FP sum was selected for a subsequent test of a sample of study genomic DNAs that had been extracted from whole blood. This screen of two 96-well plates evaluated 151 subjects (3 present in triplicate), 5 negative controls, and 30 synthetic targets (10 of each homozygote and 10 of the mixed/heterozygote). The latter were especially helpful for establishing AA, AB, and BB cluster positions and assay performance of rare SNPs (versus testing a novel assay of uncertain performance on genomic DNAs of unknown genotype). Of the 98 designed and tested assays, 85 yielded clean genotypes in study DNAs (an 87% assay conversion rate).

We proceeded to production genotyping with 77 of these SNP and indel assays (the subset that proved necessary for our work) on 2202 DNA samples to generate ∼170 000 total genotypes. We included as controls 67 duplicate genomic DNA pairs, observing two mismatched genotype calls (estimated error rate 0.0004). Independent of the SNuPE assays, we also genotyped the same DNA samples by Illumina Infinium MEGA EX array. Note that an array survey is more appropriate for assessment of genetic ancestry than customized assay of specific, required variants. Although not by our design, genotypes of 12 of the 77 variants assayed by SNuPE were also generated by the array, enabling comparison of genotype calls from an orthogonal method. One was errantly monomorphic by array. The remaining 11 SNPs yielded 17 346 duplicate genotypes with 43 discrepancies, a discrepancy rate of 0.003. These data support an accuracy for the SNuPE assay in line with that of other production genotyping approaches.

Six of the SNuPE assays that were genotyped in production had FP sums in assay development stages that we recognized could be improved by altering extension primer Tm. In the course of production genotyping, we evaluated extension primer Tm as an additional assay variable with potential to improve specific incorporation. We optimized six assays by evaluating and choosing higher Tm extension primers ( Fig. 9). Overall, extension primer Tm’s of successful assays ranged from 52.6°C to 73.1°C, averaging 58.1°C. We estimate the optimal extension primer Tm design goal to be between 60°C and 65°C.

Effect of extension primer Tm on FP sum. For each variant, the FP sum of the initial and of the optimized Tm is presented as an extended line.

Effect of extension primer Tm on FP sum. For each variant, the FP sum of the initial and of the optimized Tm is presented as an extended line.

A significant proportion of specific required variants typically fail to successfully convert for assay by any given alternative method. More than one approach is often necessary. As an independent example, among a set of 26 SNPs for which we had previously sought TaqMan assays, half were available predesigned and half required custom design. Among the custom set, six failed design, one passed design but failed actual assay, and the remaining six had good performance. Thus, 19 of 26 (73%) successfully converted for TaqMan assay, with an estimated error rate of 0.004 (four genotype mismatches among 1,139 duplicate genotype pairs).


Primer Design using Software

A number of primer design tools are available that can assist in PCR primer design for new and experienced users alike. These tools may reduce the cost and time involved in experimentation by lowering the chances of failed experimentation.

Primer Premier follows all the guidelines specified for PCR primer design. Primer Premier can be used to design primers for single templates, alignments, degenerate primer design, restriction enzyme analysis. contig analysis and design of sequencing primers.

The guidelines for qPCR primer design vary slightly. Software such as AlleleID and Beacon Designer can design primers and oligonucleotide probes for complex detection assays such as multiplex assays, cross species primer design, species specific primer design and primer design to reduce the cost of experimentation.

PrimerPlex is a software that can design primers for Multiplex PCR and multiplex SNP genotyping assays.


Methods and materials

C. elegans strains were cultured using standard methods 1 . Strains used to initiate this study are: CB1033 che-2(e1033), CB3329 che-10(e1809), IW523 che-10(iw109) daf-3(iw108), CB3241 clr-1(e1754), CB1376 daf-3(e1376), VC20208 without daf-3(gk269916), VC20379 daf-3(iw108), RB2589 daf-3(ok3610), SD378 dpy-17(e164) unc-79(e1068) / mpk-1(ga117), JN554 dyf-11(pe554), PR813 osm-5(p813), VC40961 rund-1(gk901813), MT7554 sqv-3(n2842) unc-69(e587)/qC1, MT9647 unc-29(e1072) sqv-5(n3039) / hT2, PR691 tax-2(p691) che-2(iw107), RB1546 tmc-1(ok1859), VC40425 tmc-1(gk631913), CB4856 Hawaiian HA wild isolate, and N2 wild-type strain. Additional strains were made using these strains.

The i40-699 indels from WormBase 37 version WS256 natural variant data were extracted using custom R 38 scripts in a separate study 39 . We also examined the WormBase version WS276 data to ensure that WS256 data is up-to-date. Using additional custom R scripts, we identified the nearest gene corresponding to each i40-699 indel along with the genetic and physical position of the gene using WormBase annotated gene dataset. A list of 11,556 annotated i40-699 indels are in Supplementary Table S1 with the physical position and the size of the indels, nearest gene with their physical and genetic position, frequency of appearance among the 40 wild isolates, and wild isolates with the i40-699 indel.

In designing primers, we aimed for uniform PCR condition and easy distinction of N2 and CB4856 HA DNA. For example, we chose i40-699 indels with longer N2 DNA than CB4856 for easy distinction. For identical annealing temperature, optimum melting temperature Tm was set at 60 ଌ. For identical elongation time, we kept the sizes of N2 and CB4856 products within a narrow range. With the final primer set, N2 product sizes are between 500 and 1500 bp whereas CB4856 product sizes are between 400 and 1300 bp. For easier separation of PCR products, we usually picked indels of >� bp length change, but we picked few indels of <� bp length change to have a representative i40-699 indel in most megabase (Mb) intervals. Otherwise, we picked i40-699 indels arbitrarily. To minimize confusing PCR products amplified from another part of the genome, primer-BLAST 40 was performed initially using the six C. elegans chromosome sequences as the template with specificity check against the C. elegans non-redundant (nr) nucleotide database. Additional primers were selected using primer-BLAST again or using primer3 41 with the appropriate cosmid, fosmid or YAC clone sequence, and we tested the primers until a satisfactory primer pair was identified. A total of 584 primers were tested, and the best primer pairs are listed in Supplementary Table S2 with expected sizes of the PCR products, PCR success rates under two different conditions, annotations of the position in 96-well plate where applicable, and wild isolates with the i40-699 indel.

Working stocks of premixed primer pairs were stored in 96-well plates frozen because long-term storage at 4 ଌ led to evaporation and possible degradation. Unless indicated otherwise, adult hermaphrodites were lysed individually in 10 μl of 1 × lysis buffer (40 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.45% IGEPAL, 0.45% Tween 20) with proteinase K added prior to lysis at 60 μg/ml final concentration. Lysis was performed at 65 ଌ for 1 h with inactivation at 95 ଌ for 15 min using 8-strip tubes. Lysed worms were often mixed or pooled, and combined samples were often diluted with water or 1 × lysis buffer lacking proteinase K. PCR was performed in 25 µl volume using standard conditions without detergent for Taq polymerase. Either 8-strip tubes or 96-well plates were used for PCR with aluminum foil seal for 96-well plates to reduce evaporation. The PCR temperature cycler condition was: 5′ 95 ଌ, 35 cycles of (30″ 95 ଌ, 30″ 60 ଌ, 2′ 72 ଌ), 5′ 72 ଌ. PCR products were visualized after separation by electrophoresis for 40 min at 120 V on 2% agarose gel with 

𠂑.5 l of Tris�tate-EDTA running buffer unless indicated otherwise. Recurring poor PCR results were usually rectified by new preparation of 1 × lysis buffer and discarding old stocks of 1 × lysis buffer, which could become less effective after many months.

To obtain F2 mutants for mapping, CB4856 males were mated with mutant hermaphrodites of interest. Next, F1 heterozygous hermaphrodites were placed in new plates as L4 larvae to reproduce F2 progeny by self-fertilization. Finally, F2 homozygous mutants were identified by their mutant phenotype, which could be clear (Clr) and blistering body 42 , squashed vulva (Sqv) at L4 larval stage 43 and associated sterility with a stereotypical morphology of embryos incapable of cytokinesis 44 , or a temperature-dependent constitutive dauer mutant phenotype 45 . Collection of temperature-dependent dauer mutants involved placing a mixed population of embryos and L1 larvae at 28 ଌ for 10 days in the presence of plenty of E. coli bacteria serving as food prior to a return to 20 ଌ to allow mutant dauer survivors to become mutant fertile adults.

Complementation tests using the temperature-dependent constitutive dauer mutant phenotype were performed using a few different approaches. For iw108 located in chromosome X, either daf-3(ok3610) or dyf-11(pe554) mutant hermaphrodites were mated with N2 males, and F1 hemizygous mutant XO males were mated with either daf-3(iw108) or dpy-17(e164) unc-79(e1068) daf-3(iw108) mutant hermaphrodites. Mixed populations of F2 progeny were moved to 28 ଌ for 10 days and subsequently were moved to 20 ଌ, and non-Dpy non-Unc fertile adult hermaphrodites were sought in the presence of alive males. Dauer survivors that became adult males were used to assess successful mating with the absence of fertile hermaphrodites indicating no complementation. With tests using daf-3(iw108) hermaphrodite mutants without dpy-17 or unc-79 mutations, dauer survivors that became fertile adult hermaphrodites were examined using PCR to confirm the presence or absence of the mutations of interest. Similar approach was used with iw107 also located in chromosome X. Here, one of che-2(e1033), daf-3(ok3610), dyf-11(pe554), rund-1(gk901813), osm-5(p813), or iw107 mutant hermaphrodites were mated with N2 males, and F1 hemizygous mutant males were mated with one of che-2(iw107), dpy-17(e164) unc-79(e1068) che-2(iw107), dpy-17(e164) unc-79(e1068) che-2(e1033), dpy-17(e164) unc-79(e1068) dyf-11(pe554), or dpy-17(e164) unc-79(e1068) osm-5(p813) hermaphrodites. A different approach was used with iw109 located in chromosome II. Here, che-10(e1809) mutant hermaphrodites were mated with N2 males, and heterozygous F1 males were mated with either che-10(iw109) or dpy-17(e164) unc-79(e1068) che-10(iw109) mutant hermaphrodites. After 10 days at 28 ଌ, the presence of dauer survivors that became males was used as evidence of complementation.

Whole genome sequencing of che-10(iw109) mutant strain was performed with Novogene (en.novogene.com), using their Plant and Animal Whole Genome Sequencing service with 2 Gb data output for 20 ×𠂜overage of the genome by paired-end 150 bp sequencing. Sanger sequencing was performed with Genewiz (www.genewiz.com).


MCAT - MOLECULAR BIOLOGY

1. The double stranded DNA must separate or unwind
• DNA gyrase (class II topoisomerase) is responsible for uncoiling the DNA ahead of the replication fork
• DNA helicase is responsible for unwinding the DNA at the replication fork
• Single-strand binding protein (SSB) is responsible for keeping the DNA unwound after the helicase. SSBs stabilize single-stranded DNA by binding to it.

2. Next, DNA is synthesized to be complementary to the newly unwound/separated DNA. All biological DNA synthesis occurs from the 5' to the 3' end.
• Primase gets this started by laying down a short RNA primer on the unwound DNA. The primer is made of RNA, but is complementary to the DNA sequence. Later, this RNA is replaced with DNA
• DNA polymerase then takes over and makes DNA that is complementary to the unwound DNA
• DNA synthesis occurs on both strands of the unwound DNA. The synthesis that proceeds in the direction of the replication fork is the leading strand. The synthesis that proceeds in the opposite direction to the replication fork is the lagging strand. The lagging strand contains Okazaki fragments.


Abstract

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5′rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.


For more information about SNPs:

An audio definition of SNPs is available from the National Human Genome Research Institute’s Talking Glossary of Genetic Terms.

How scientists locate SNPs in the genome is explained by the University of Utah Genetic Science Learning Center.

For people interested in more technical data, several databases of known SNPs are available: