CN112176046A - Amplification method for analyzing CGG repeat number of upstream untranslated region of FMR1 gene - Google Patents

Amplification method for analyzing CGG repeat number of upstream untranslated region of FMR1 gene Download PDF

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CN112176046A
CN112176046A CN202011096517.7A CN202011096517A CN112176046A CN 112176046 A CN112176046 A CN 112176046A CN 202011096517 A CN202011096517 A CN 202011096517A CN 112176046 A CN112176046 A CN 112176046A
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deaminase
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杜利军
朱应竹
谢正顺
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Huadu District People's Hospital Of Guangzhou
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Huadu District People's Hospital Of Guangzhou
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Abstract

The invention discloses an amplification method for analyzing the CGG repeat number of an upstream untranslated region of an FMR1 gene, which comprises the following steps: (1) constructing a compound of three TALEs covalently coupled with deaminase; (2) expressing the three complexes in escherichia coli; (3) testing the protein activity expressed by the three complexes; (4) extracting target DNA; (5) adding a reaction buffer solution and recombinant TALE-deaminase into the target DNA for reaction; (6) amplifying DNA after deamination reaction by PCR; (7) the sequence of the PCR amplification product was determined by Sanger sequencing. The invention belongs to the technical field of medical inspection science, and discloses a method for analyzing the duplication number of CGG in an upstream untranslated region of an FMR1 gene.

Description

Amplification method for analyzing CGG repeat number of upstream untranslated region of FMR1 gene
Technical Field
The invention belongs to the technical field of medical inspection science, and particularly relates to an amplification method for analyzing the number of CGG repeats in an upstream untranslated region of an FMR1 gene.
Background
Fragile X Syndrome (FXS) is an X-chromosome linked genetic disease. It is typically characterized by moderate to severe intellectual impairment, giant testis (50% of patients have 30-50 ml of testis volume, 20ml of normal value), big ear, language disorder, mental retardation, and Intelligence Quotient (IQ) of 0-50. The patients are mostly male and the male symptoms are severe. Women have two X chromosomes which are mostly carriers, wherein 2/3 have normal intelligence and 1/3 has slight mental retardation. In the Fragile X syndrome family, diseases related to the causes of the Fragile X syndrome may occur, including Fragile X-Associated tremor ataxia syndrome (FXTAS), Fragile X-Associated Primary Ovarian Insufficiency (FXPOI), and the like.
Fragile X syndrome is caused by the absence and deletion of the Familial Mental Retardation Protein (FMRP), more than 95% of Fragile X syndrome is caused by the extension of CGG repeats in the untranslated region upstream of the FMR1 gene, and the extension of large segments of CGG leads to hypermethylation in this region and thus to inhibition of transcription of the FMR1 gene. Less than 5% of patients are caused by FMRP protein dysfunction due to deletion mutations or point mutations in the FMR1 gene.
The detection of the number of CGG repeats can be used for diagnosing FXS, the sensitivity of the index is more than 99%, and the specificity is 100%. CpG islands are not methylated when the (CGG) n repeat number is 5-44, (CGG) n repeat is stable, FMRP expression is normal, and no pathogenicity exists; CpG islands are not methylated when the number of (CGG) n repeats is 45-54, (CGG) n repeats are uncertain in stability, FMRP expression is normal and non-pathogenic; when the number of (CGG) n repeats is 55-200, called premutations (pretutations), CpG islands are not methylated, (CGG) n repeats are easily expandable, when FMRP transcription increases, translation decreases, Fragile X-Associated tremator ataxia syndrome (FXTAS) and Fragile X-Associated Primary Ovarian Insufficiency (FXPOI) occur; when the number of (CGG) n repeats is greater than 200, called full mutations, CpG islands are methylated, and (CGG) n repeats are easily expanded and FMRP is not expressed, then men will show Fragile X Syndrome (FXS) and women will be carriers. 1/4,000 men were full mutations, with 0.61% full mutations and 1.7% pre-mutations in women. The genetic pattern is X-linked and may be expanded by the next generation.
Detection of CGG repeat numbers is diagnostic rather than predictive for males because the penetrance of males is 100%. Full mutations may be diagnosable in women, but less than 50% of women carrying full mutations exhibit mental retardation, which may manifest avoidant personality and stereotyped movements. The identification of a premutation in asymptomatic men and women is predictive, since FXPOI and FXTAS are not all dominant, related to age and the number of repeats in CGG.
Carrier screening and neonatal screening in populations is not currently recommended and can only be conducted as a scientific research. Prenatal screening can be performed using amniotic fluid or villous puncture. It should be noted that methylation has not occurred at this point. Because of somatic mutations, full mutations may show streaks, which may vary in size, which is inconsistent with blood samples. Chimeras of trophoblast and somatic cells are present, and amniocentesis may be a full mutation if villous puncture shows a premutation. The shortest number of repeats found to be capable of expanding to full mutations in the next generation is 56, and pregnant women carrying this number of repeats need to be prenatally diagnosed with possible chimeras, including chimeras of the number of repeats and methylated chimeras.
The detection method commonly used at present comprises the following steps:
DNA hybridization (Southern blot): small pro-mutations are easily detected when the normal fragment is small and/or the electrophoretic mobility is long, whereas large/diffuse full mutations are more easily detected when the normal fragment is large and/or the electrophoretic mobility is short. In female samples, it should be noted that the phenotype of two different sized normal repeats on DNA hybridization may be consistent with the phenotype on DNA hybridization carrying one normal mutation and a pre-mutation. The fully mutated band may be diffuse and weak and the signal-to-noise ratio to be tested is good. In males, the full mutation is unlikely to be overlooked because the normal band would be absent or light in color (in the case of size chimeras), but if the background interference is large, the full mutation may be missed in the female sample. If enzymes sensitive to methylation are used for digestion in DNA hybridization, this may help: 1) if the repeat size is well around a threshold, e.g., 200, it can help to distinguish between pre-mutations and full mutations; 2) rare methylated chimera individuals are detected; 3) abnormal chromosome types, e.g. 45, X or 47, XXY, are detected. The methylation level of the RMR1 gene cannot be used to assess the severity of disease in fetuses and newborns, whether the DNA is from amniotic fluid, villi, or blood.
2. Polymerase Chain Reaction (Polymerase Chain Reaction, PCR): PCR labeled with the radioisotope P-32 and electrophoresed on polyacrylamide gel, or fluorescence labeled primers and subjected to capillary electrophoresis. If there is a polymorphism in the primer binding region, the PCR detection fails, but no polymorphic site affecting the binding of the usual primer has been found so far. Product size standards are necessary. Capillary Electrophoresis (CE) may use fluorescently labeled standards and polyacrylamide electrophoresis may use standard molecular scales. Conventional PCR strategy amplification cannot amplify large repeats and therefore cannot distinguish between a normally homozygous woman and a heterozygous woman carrying a normal gene and a non-amplifiable gene. Similarly, a sample may show only the premutation if it is a chimera of the premutation and the full mutation. Even if the PCR strategy is able to detect large repeats, the amplification will be biased towards small repeats in all samples containing multiple repeats. For example, chimeras in female samples, form heteroduplexes (heteroduplexes) when the female sample and chimeras are amplified. Ordinary PCR amplification is not affected by methylation, but PCR can be adjusted to detect methylation. When a PCR strategy is used to detect full mutations, the hot spots of deletion mutations in the CGG repeats need to be noted. If the primer is exactly in the deletion mutation hotspot, amplification will fail; if prior to the deletion mutation, a significant size chimera results. The three-primer PCR method (TRP-PCR) can improve the amount of amplified full mutation, but the amplification product is diffuse, and the normal repetition can suppress the repetition of expansion, which can influence the detection accuracy.
Deaminase can catalyze the hydrolytic deamination of cytosine and 5-methylcytosine to form uracil and thymine, converting the CG base to the TA base. This property of deaminase may be useful in the fields of gene therapy and the like.
Transcription Activator-Like Effectors (TALEs) are Transcription Activator-Like Effectors secreted by the plant pathogen Xanthomonas (Xanthomonas) and recognize DNA sequences. The DNA binding domain of the TALE protein is formed by connecting amino acid sequence modules capable of recognizing single nucleotide bases in series, and the amino acid sequence has a determined corresponding relation with the nucleic acid sequence of a target site. The sequence module of TALE can be used for assembling recombinant protein which is specifically combined with any DNA sequence, thereby achieving the purpose of identifying the DNA sequence.
Disclosure of Invention
In view of the above, in order to overcome the defects that the amount of total mutation is suppressed by normal repetition when CGG is repeated by PCR (TRP-PCR) amplification and the amplification product is diffuse, the invention provides a method for analyzing the number of CGG repeats in an upstream untranslated region of an FMR1 gene, which can detect the number of CGG repeats related to fragile X syndrome with higher signal-to-noise ratio, particularly the previous mutation and the total mutation with larger length, and can diagnose the fragile X syndrome more reliably.
The technical scheme adopted by the invention is as follows: the amplification method for analyzing the number of CGG repeats in the upstream untranslated region of the FMR1 gene comprises the following steps:
(1) constructing a compound of three Transcription Activator Like Effectors (TALE) and deaminase which are covalently coupled, wherein the TALE and the deaminase are flexibly connected by using 15 glycines, and the three compounds are CGG-TALE-downstream deaminase, TAA-TALE-downstream deaminase and CGG-TALE-upstream deaminase respectively;
(2) expressing the three complexes in escherichia coli;
(3) testing the activity of the protein expressed by the three compounds, mixing the three compounds after the activity is tested to obtain recombinant TALE-deaminase, and adjusting the activity of each compound to be 1U/mu L, wherein 1U is defined as the enzyme activity of converting 50 percent of 1ug of guanine into adenine in a 10uL reaction system at 37 ℃ within 60 minutes;
(4) extracting target DNA, and adjusting the concentration of the target DNA to be between 100 and 500 ng/. mu.L;
(5) adding 10 mu L of reaction buffer solution and 1 mu L of recombinant TALE-deaminase into 10 mu L of target DNA, reacting for 4 hours AT 37 ℃, deaminating GC base in a specific region in the target DNA in a directional manner to convert the GC base into AT base, and inactivating for 10 minutes AT 95 ℃ after the reaction is finished;
(6) amplifying DNA after deamination reaction by PCR;
(7) determining the sequence of the PCR amplification product by a Sanger sequencing method;
(8) and (6) analyzing the result.
By adopting the steps, the invention has the following beneficial effects: according to the scheme, a TALE-deaminase is utilized to process a sample, and GC bases in a specific region in target DNA are deaminated and converted into AT bases in a directional mode, so that the GC content in the sample is reduced, isometric amplification and sequencing of the gene are feasible, the CGG repetition number of the upstream untranslated region of the FMR1 gene is analyzed more accurately, and the method is used as a basis for diagnosing fragile X syndrome and related genetic diseases.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram showing the method of amplifying the number of CGG repeats in the upstream untranslated region of FMR1 gene according to the present invention;
FIG. 2 shows the sequencing result of male (CGG) with n-repeats of 20 according to the amplification method of the present invention for analyzing the number of CGG repeats in the upstream untranslated region of FMR1 gene;
FIG. 3 shows the sequencing result of male (CGG) with n-repeat number of 51 according to the amplification method of the present invention for analyzing the CGG-repeat number of the upstream untranslated region of FMR1 gene;
FIG. 4 shows the sequencing result of 78 for male (CGG) n repeat number according to the amplification method for analyzing the CGG repeat number of the upstream untranslated region of FMR1 gene of the present invention;
FIG. 5 shows the sequencing result of male (CGG) with n-repeat number 217 according to the amplification method of the present invention for analyzing the CGG-repeat number of the untranslated region upstream of FMR1 gene.
Wherein, 1, deaminase, 2, target DNA before deamination, 3, transcription activator like effector, 4, partial deamination DNA, 5 and DNA after deamination reaction.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to an amplification method for analyzing the number of CGG repeats in an upstream untranslated region of an FMR1 gene, which comprises the following steps:
(1) constructing a compound of three Transcription Activator Like Effectors (TALEs) and deaminase which are covalently coupled, wherein the TALEs and the deaminase are flexibly connected by using 15 glycines, and the three compounds are CGG-TALE-downstream deaminase, TAA-TALE-downstream deaminase and CGG-TALE-upstream deaminase respectively;
(2) expressing the three complexes in escherichia coli;
(3) testing the activity of the protein expressed by the three compounds, mixing the three enzymes after the determination to obtain the recombinant TALE-deaminase, and adjusting the activity of each enzyme to 1U/mu L;
(4) extracting target DNA, and adjusting the concentration of the target DNA to be between 100 and 500 ng/. mu.L;
(5) adding 10 mu L of reaction buffer solution and 1 mu L of recombinant TALE-deaminase into 10 mu L of target DNA, reacting for 4 hours AT 37 ℃, directionally changing GC base of a specific region in the target DNA into AT base, and inactivating for 10 minutes AT 95 ℃ after the reaction is finished;
(6) amplifying DNA after deamination reaction by PCR;
(7) determining the sequence of the PCR amplification product by adopting a Sanger sequencing method;
(8) and (6) analyzing the result.
Example (b): the detection of the number of CGG repeats in the upstream region of the upstream untranslated region of the human FMR1 gene was carried out by an amplification method for analyzing the number of CGG repeats in the upstream untranslated region of the FMR1 gene.
(I) preparing experimental components and experimental tools
(1) Preparing the components required by the experiment:
preparing the recombinant TALE-deaminase: cloning a gene sequence of TALE-deaminase to an escherichia coli expression vector PET28 a;
wherein, CGG-TALE-downstream deaminase expression sequence (containing an initiation codon and a stop codon):
ATGCATGGCCTGACTCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACTCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACTCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACTCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACTCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAAGGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGATGTAGAAAAATTAATTGCTGAATCGAAAAAAGCACGGGAGCAAGCGTATGTCCCGTATTCAAAATTCCCAGTTGGAGCGGCGTTGTTAGCCGAAGATGGGACTATCTATCACGGCTGTAATATTGAAAACTCCGCTTATAGCATGACCAATTGTGCAGAACGAACAGCATTTTTTAAAGCTGTTTCCGACGGCGTACGTAGCTTCAAAGCGCTTGCAGTAGTTGCAGATACAGAAGGACCTGTTTCCCCGTGTGGAGCATGCAGACAAGTAATTGCGGAGTTTTGTAATGGCTCGATGCCTGTCTACCTCACGAACCTAAAAGGGGACATTGAAGAAACAACTGTAGCAAAATTATTACCAGGCGCATTTTCAAAGGAGGATCTTTCATATGCAGCAGAACAATAA
ATT-TALE-downstream deaminase expression sequence (containing start and stop codons):
ATGCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGATGTAGAAAAATTAATTGCTGAATCGAAAAAAGCACGGGAGCAAGCGTATGTCCCGTATTCAAAATTCCCAGTTGGAGCGGCGTTGTTAGCCGAAGATGGGACTATCTATCACGGCTGTAATATTGAAAACTCCGCTTATAGCATGACCAATTGTGCAGAACGAACAGCATTTTTTAAAGCTGTTTCCGACGGCGTACGTAGCTTCAAAGCGCTTGCAGTAGTTGCAGATACAGAAGGACCTGTTTCCCCGTGTGGAGCATGCAGACAAGTAATTGCGGAGTTTTGTAATGGCTCGATGCCTGTCTACCTCACGAACCTAAAAGGGGACATTGAAGAAACAACTGTAGCAAAATTATTACCAGGCGCATTTTCAAAGGAGGATCTTTCATATGCAGCAGAACAATAA
ATT-TALE-upstream deaminase expression sequence (containing start codon and stop codon):
ATGGATGTAGAAAAATTAATTGCTGAATCGAAAAAAGCACGGGAGCAAGCGTATGTCCCGTATTCAAAATTCCCAGTTGGAGCGGCGTTGTTAGCCGAAGATGGGACTATCTATCACGGCTGTAATATTGAAAACTCCGCTTATAGCATGACCAATTGTGCAGAACGAACAGCATTTTTTAAAGCTGTTTCCGACGGCGTACGTAGCTTCAAAGCGCTTGCAGTAGTTGCAGATACAGAAGGACCTGTTTCCCCGTGTGGAGCATGCAGACAAGTAATTGCGGAGTTTTGTAATGGCTCGATGCCTGTCTACCTCACGAACCTAAAAGGGGACATTGAAGAAACAACTGTAGCAAAATTATTACCAGGCGCATTTTCAAAGGAGGATCTTTCATATGCAGCAGAACAAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGACATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACATTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACTAA
for each protein, after sequencing the plasmid to determine the sequence, transformed into BL21(DE3) type Escherichia coli, then inoculated into LB medium, at 37 degrees C and 220rpm rotation speed oscillation culture to OD value of 0.5-0.6, adding a final concentration of 50ug/mL isopropyl-beta-D-thiogalactoside (IPTG), at 37 degrees C and 220rpm speed oscillation culture for 1.5 hours, 5000g centrifugal 10 minutes, abandon the supernatant, using PBS heavy suspension, 5000g centrifugal 10 minutes, using PBS heavy suspension, adding a final concentration of 5 u L/mL lysozyme, room temperature for 2 hours, ultrasonic cell breaker ultrasonic treatment for 30 minutes, nickel column purification of the protein, and using the ultraviolet method for determination of the concentration of the collected protein. And adjusting the activity to 1U/mu L;
(2) a metal bath;
(3) enzyme solution: kod-plus DNA polymerase, glycerol, tris, sodium chloride;
(4) PCR reaction solution I: dNTPs, tris, sodium chloride, upstream primer: 5' -AGCGCATCGCACTCAGGGTGTGACGGAGGCGCCGCTG, downstream primer: 5' -CGAGCTCACGCCTGTGAGCCTCCATCTTCTCTTCAGCCCTGCTA;
(5) PCR reaction solution II: dNTPs, MgSO4, tris, sodium chloride, upstream primer: 5' -AGCGCATCGCACTCAGGGT, downstream primer: 5' -CGAGCTCACGCCTGTGAGC.
(6) A polyethylene PCR reaction tube;
(7) equipment: ABI7500 fluorescent quantitative PCR instrument.
(II) the experimental operation steps are as follows:
1) eluting the target DNA with purified water, and adjusting the concentration of the target DNA to be between 100 and 500 ng/. mu.L;
2) adding 10 mu L of reaction buffer solution and 1U of TALE-deaminase into 10 mu LDNA, reacting for 2 hours at 37 ℃, and inactivating for 10 minutes at 95 ℃ after the reaction is finished;
3) the first step of PCR reaction: taking 1 mu L of the inactivated sample in the step 2, placing the sample in a PCR tube, and adding 23.5 mu L of PCR reaction solution I and 0.5 mu L of enzyme solution, wherein the reaction procedure is as follows: 94 ℃ for 1 min; 94 ℃, 15s, 60 ℃, 60s, 26 circles; at 20 deg.C for 1 min;
4) the second step of PCR reaction: taking 1 μ L of the sample in the step 3, placing the sample in a PCR tube, and adding 23.5 μ L of the PCR reaction solution II and 0.5 μ L of the enzyme solution, wherein the reaction procedure is as follows: 94 ℃ for 1 min; 94 ℃, 15s, 60 ℃, 60s, 26 circles; at 20 deg.C for 1 min;
5) performing Sanger sequencing on the PCR amplification product;
6) and (6) analyzing the result.
FIG. 1 is a schematic diagram showing the amplification method of the present invention for analyzing the number of CGG repeats in the upstream untranslated region of FMR1 gene, the amplification method being as follows: the GC bases of a specific region in the target DNA are deaminated to AT bases using TALE-deaminase.
The experimental results are shown in FIGS. 2 to 5:
FIG. 2: sequencing results for male (CGG) n repeat number 20: GTGACGGAGGCGCCGCTGCCAGGGGGCGTGCAATAATATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATTAAATCTCGAGCGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCCGAGCTCACGCCTGTGAGCCTCCATCTTCTCTTCAGCCCTGCTA are provided.
FIG. 3: sequencing results for male (CGG) with a repeat number of n of 51: GTGACGGAGGCGCCGCTGCCAGGGGGCGTGCAATAATATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATTAAATCTCGAGCGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCCGAGCTCACGCCTGTGAGCCTCCATCTTCTCTTCAGCCCTGCTA are provided.
FIG. 4: sequencing result for male (CGG) n repeat number 78: GTGACGGAGGCGCCGCTGCCAGGGGGCGTGCAATAATATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATTAAATCTCGAGCGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCCGAGCTCACGCCTGTGAGCCTCCATCTTCTCTTCAGCCCTGCTA are provided.
FIG. 5: sequencing result for male (CGG) with n-repeat number 217: GTGACGGAGGCGCCGCTGCCAGGGGGCGTGCAATAATATAATAATAATAATAATAATAATAATAATAAAAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAAAAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATTAAATCTCGAGCGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCCGAGCTCACGCCTGTGAGCCTCCATCTTCTCTTCAGCCCTGCTA are provided.
As shown in FIGS. 2-5, the analysis can be more clearly performed by the method for samples with less than 218 CGG repeats.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Guangzhou city flower city district people hospital
<120> amplification method for analyzing CGG repeat number in untranslated region upstream of FMR1 Gene
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1986
<212> PRT
<213> CGG-TALE-downstream deaminase expression sequence (containing start codon and stop codon)
<400> 1
Ala Thr Gly Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Thr Cys
1 5 10 15
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
20 25 30
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Cys Ala Cys Gly Ala Thr
35 40 45
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
50 55 60
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
65 70 75 80
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
85 90 95
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
100 105 110
Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
115 120 125
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
130 135 140
Ala Ala Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
145 150 155 160
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
165 170 175
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
180 185 190
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys
195 200 205
Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala
210 215 220
Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys
225 230 235 240
Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly Cys Gly
245 250 255
Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala
260 265 270
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly
275 280 285
Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys
290 295 300
Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
305 310 315 320
Thr Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
325 330 335
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Cys Ala Cys Gly
340 345 350
Ala Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
355 360 365
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
370 375 380
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
385 390 395 400
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
405 410 415
Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala
420 425 430
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
435 440 445
Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys Ala Ala
450 455 460
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
465 470 475 480
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
485 490 495
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
500 505 510
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
515 520 525
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
530 535 540
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly
545 550 555 560
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
565 570 575
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
580 585 590
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
595 600 605
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
610 615 620
Ala Cys Thr Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
625 630 635 640
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Cys Ala
645 650 655
Cys Gly Ala Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
660 665 670
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
675 680 685
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
690 695 700
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr
705 710 715 720
Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys
725 730 735
Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys
740 745 750
Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys
755 760 765
Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala
770 775 780
Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr
785 790 795 800
Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly
805 810 815
Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys
820 825 830
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
835 840 845
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly
850 855 860
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
865 870 875 880
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
885 890 895
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
900 905 910
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
915 920 925
Thr Gly Ala Cys Thr Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
930 935 940
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
945 950 955 960
Cys Ala Cys Gly Ala Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
965 970 975
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
980 985 990
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
995 1000 1005
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys
1010 1015 1020
Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala
1025 1030 1035 1040
Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys
1045 1050 1055
Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly Cys Gly
1060 1065 1070
Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala
1075 1080 1085
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly
1090 1095 1100
Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys
1105 1110 1115 1120
Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
1125 1130 1135
Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
1140 1145 1150
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala
1155 1160 1165
Ala Gly Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
1170 1175 1180
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
1185 1190 1195 1200
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
1205 1210 1215
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
1220 1225 1230
Cys Cys Thr Gly Ala Cys Thr Cys Cys Gly Gly Ala Cys Cys Ala Ala
1235 1240 1245
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
1250 1255 1260
Gly Cys Cys Ala Cys Gly Ala Thr Gly Gly Cys Gly Gly Cys Ala Ala
1265 1270 1275 1280
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
1285 1290 1295
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
1300 1305 1310
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
1315 1320 1325
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
1330 1335 1340
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
1345 1350 1355 1360
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Gly
1365 1370 1375
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
1380 1385 1390
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
1395 1400 1405
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
1410 1415 1420
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
1425 1430 1435 1440
Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
1445 1450 1455
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala
1460 1465 1470
Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
1475 1480 1485
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
1490 1495 1500
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
1505 1510 1515 1520
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Gly Gly Ala
1525 1530 1535
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly
1540 1545 1550
Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly
1555 1560 1565
Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Thr Gly Thr Ala
1570 1575 1580
Gly Ala Ala Ala Ala Ala Thr Thr Ala Ala Thr Thr Gly Cys Thr Gly
1585 1590 1595 1600
Ala Ala Thr Cys Gly Ala Ala Ala Ala Ala Ala Gly Cys Ala Cys Gly
1605 1610 1615
Gly Gly Ala Gly Cys Ala Ala Gly Cys Gly Thr Ala Thr Gly Thr Cys
1620 1625 1630
Cys Cys Gly Thr Ala Thr Thr Cys Ala Ala Ala Ala Thr Thr Cys Cys
1635 1640 1645
Cys Ala Gly Thr Thr Gly Gly Ala Gly Cys Gly Gly Cys Gly Thr Thr
1650 1655 1660
Gly Thr Thr Ala Gly Cys Cys Gly Ala Ala Gly Ala Thr Gly Gly Gly
1665 1670 1675 1680
Ala Cys Thr Ala Thr Cys Thr Ala Thr Cys Ala Cys Gly Gly Cys Thr
1685 1690 1695
Gly Thr Ala Ala Thr Ala Thr Thr Gly Ala Ala Ala Ala Cys Thr Cys
1700 1705 1710
Cys Gly Cys Thr Thr Ala Thr Ala Gly Cys Ala Thr Gly Ala Cys Cys
1715 1720 1725
Ala Ala Thr Thr Gly Thr Gly Cys Ala Gly Ala Ala Cys Gly Ala Ala
1730 1735 1740
Cys Ala Gly Cys Ala Thr Thr Thr Thr Thr Thr Ala Ala Ala Gly Cys
1745 1750 1755 1760
Thr Gly Thr Thr Thr Cys Cys Gly Ala Cys Gly Gly Cys Gly Thr Ala
1765 1770 1775
Cys Gly Thr Ala Gly Cys Thr Thr Cys Ala Ala Ala Gly Cys Gly Cys
1780 1785 1790
Thr Thr Gly Cys Ala Gly Thr Ala Gly Thr Thr Gly Cys Ala Gly Ala
1795 1800 1805
Thr Ala Cys Ala Gly Ala Ala Gly Gly Ala Cys Cys Thr Gly Thr Thr
1810 1815 1820
Thr Cys Cys Cys Cys Gly Thr Gly Thr Gly Gly Ala Gly Cys Ala Thr
1825 1830 1835 1840
Gly Cys Ala Gly Ala Cys Ala Ala Gly Thr Ala Ala Thr Thr Gly Cys
1845 1850 1855
Gly Gly Ala Gly Thr Thr Thr Thr Gly Thr Ala Ala Thr Gly Gly Cys
1860 1865 1870
Thr Cys Gly Ala Thr Gly Cys Cys Thr Gly Thr Cys Thr Ala Cys Cys
1875 1880 1885
Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Ala Ala Ala Ala Gly Gly
1890 1895 1900
Gly Gly Ala Cys Ala Thr Thr Gly Ala Ala Gly Ala Ala Ala Cys Ala
1905 1910 1915 1920
Ala Cys Thr Gly Thr Ala Gly Cys Ala Ala Ala Ala Thr Thr Ala Thr
1925 1930 1935
Thr Ala Cys Cys Ala Gly Gly Cys Gly Cys Ala Thr Thr Thr Thr Cys
1940 1945 1950
Ala Ala Ala Gly Gly Ala Gly Gly Ala Thr Cys Thr Thr Thr Cys Ala
1955 1960 1965
Thr Ala Thr Gly Cys Ala Gly Cys Ala Gly Ala Ala Cys Ala Ala Thr
1970 1975 1980
Ala Ala
1985
<210> 2
<211> 1986
<212> PRT
<213> ATT-TALE-downstream deaminase expression sequence (containing start codon and stop codon)
<400> 2
Ala Thr Gly Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys
1 5 10 15
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
20 25 30
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala Thr Thr
35 40 45
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
50 55 60
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
65 70 75 80
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
85 90 95
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
100 105 110
Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
115 120 125
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
130 135 140
Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
145 150 155 160
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
165 170 175
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
180 185 190
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys
195 200 205
Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala
210 215 220
Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys
225 230 235 240
Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly
245 250 255
Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala
260 265 270
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly
275 280 285
Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys
290 295 300
Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
305 310 315 320
Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
325 330 335
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala
340 345 350
Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
355 360 365
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
370 375 380
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
385 390 395 400
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
405 410 415
Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala
420 425 430
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
435 440 445
Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala
450 455 460
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
465 470 475 480
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
485 490 495
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
500 505 510
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
515 520 525
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
530 535 540
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly
545 550 555 560
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
565 570 575
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
580 585 590
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
595 600 605
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
610 615 620
Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
625 630 635 640
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala
645 650 655
Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
660 665 670
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
675 680 685
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
690 695 700
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr
705 710 715 720
Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys
725 730 735
Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys
740 745 750
Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys
755 760 765
Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala
770 775 780
Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr
785 790 795 800
Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly
805 810 815
Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys
820 825 830
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
835 840 845
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr
850 855 860
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
865 870 875 880
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
885 890 895
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
900 905 910
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
915 920 925
Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
930 935 940
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
945 950 955 960
Ala Ala Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
965 970 975
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
980 985 990
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
995 1000 1005
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys
1010 1015 1020
Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala
1025 1030 1035 1040
Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys
1045 1050 1055
Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly
1060 1065 1070
Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala
1075 1080 1085
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly
1090 1095 1100
Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys
1105 1110 1115 1120
Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
1125 1130 1135
Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
1140 1145 1150
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly
1155 1160 1165
Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
1170 1175 1180
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
1185 1190 1195 1200
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
1205 1210 1215
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
1220 1225 1230
Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala
1235 1240 1245
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
1250 1255 1260
Gly Cys Ala Ala Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala
1265 1270 1275 1280
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
1285 1290 1295
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
1300 1305 1310
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
1315 1320 1325
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
1330 1335 1340
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
1345 1350 1355 1360
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly
1365 1370 1375
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
1380 1385 1390
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
1395 1400 1405
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
1410 1415 1420
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
1425 1430 1435 1440
Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
1445 1450 1455
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala
1460 1465 1470
Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
1475 1480 1485
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
1490 1495 1500
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
1505 1510 1515 1520
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Gly Gly Ala
1525 1530 1535
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly
1540 1545 1550
Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly
1555 1560 1565
Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Thr Gly Thr Ala
1570 1575 1580
Gly Ala Ala Ala Ala Ala Thr Thr Ala Ala Thr Thr Gly Cys Thr Gly
1585 1590 1595 1600
Ala Ala Thr Cys Gly Ala Ala Ala Ala Ala Ala Gly Cys Ala Cys Gly
1605 1610 1615
Gly Gly Ala Gly Cys Ala Ala Gly Cys Gly Thr Ala Thr Gly Thr Cys
1620 1625 1630
Cys Cys Gly Thr Ala Thr Thr Cys Ala Ala Ala Ala Thr Thr Cys Cys
1635 1640 1645
Cys Ala Gly Thr Thr Gly Gly Ala Gly Cys Gly Gly Cys Gly Thr Thr
1650 1655 1660
Gly Thr Thr Ala Gly Cys Cys Gly Ala Ala Gly Ala Thr Gly Gly Gly
1665 1670 1675 1680
Ala Cys Thr Ala Thr Cys Thr Ala Thr Cys Ala Cys Gly Gly Cys Thr
1685 1690 1695
Gly Thr Ala Ala Thr Ala Thr Thr Gly Ala Ala Ala Ala Cys Thr Cys
1700 1705 1710
Cys Gly Cys Thr Thr Ala Thr Ala Gly Cys Ala Thr Gly Ala Cys Cys
1715 1720 1725
Ala Ala Thr Thr Gly Thr Gly Cys Ala Gly Ala Ala Cys Gly Ala Ala
1730 1735 1740
Cys Ala Gly Cys Ala Thr Thr Thr Thr Thr Thr Ala Ala Ala Gly Cys
1745 1750 1755 1760
Thr Gly Thr Thr Thr Cys Cys Gly Ala Cys Gly Gly Cys Gly Thr Ala
1765 1770 1775
Cys Gly Thr Ala Gly Cys Thr Thr Cys Ala Ala Ala Gly Cys Gly Cys
1780 1785 1790
Thr Thr Gly Cys Ala Gly Thr Ala Gly Thr Thr Gly Cys Ala Gly Ala
1795 1800 1805
Thr Ala Cys Ala Gly Ala Ala Gly Gly Ala Cys Cys Thr Gly Thr Thr
1810 1815 1820
Thr Cys Cys Cys Cys Gly Thr Gly Thr Gly Gly Ala Gly Cys Ala Thr
1825 1830 1835 1840
Gly Cys Ala Gly Ala Cys Ala Ala Gly Thr Ala Ala Thr Thr Gly Cys
1845 1850 1855
Gly Gly Ala Gly Thr Thr Thr Thr Gly Thr Ala Ala Thr Gly Gly Cys
1860 1865 1870
Thr Cys Gly Ala Thr Gly Cys Cys Thr Gly Thr Cys Thr Ala Cys Cys
1875 1880 1885
Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Ala Ala Ala Ala Gly Gly
1890 1895 1900
Gly Gly Ala Cys Ala Thr Thr Gly Ala Ala Gly Ala Ala Ala Cys Ala
1905 1910 1915 1920
Ala Cys Thr Gly Thr Ala Gly Cys Ala Ala Ala Ala Thr Thr Ala Thr
1925 1930 1935
Thr Ala Cys Cys Ala Gly Gly Cys Gly Cys Ala Thr Thr Thr Thr Cys
1940 1945 1950
Ala Ala Ala Gly Gly Ala Gly Gly Ala Thr Cys Thr Thr Thr Cys Ala
1955 1960 1965
Thr Ala Thr Gly Cys Ala Gly Cys Ala Gly Ala Ala Cys Ala Ala Thr
1970 1975 1980
Ala Ala
1985
<210> 3
<211> 1986
<212> PRT
<213> ATT-TALE-upstream deaminase expression sequence (containing start codon and stop codon)
<400> 3
Ala Thr Gly Gly Ala Thr Gly Thr Ala Gly Ala Ala Ala Ala Ala Thr
1 5 10 15
Thr Ala Ala Thr Thr Gly Cys Thr Gly Ala Ala Thr Cys Gly Ala Ala
20 25 30
Ala Ala Ala Ala Gly Cys Ala Cys Gly Gly Gly Ala Gly Cys Ala Ala
35 40 45
Gly Cys Gly Thr Ala Thr Gly Thr Cys Cys Cys Gly Thr Ala Thr Thr
50 55 60
Cys Ala Ala Ala Ala Thr Thr Cys Cys Cys Ala Gly Thr Thr Gly Gly
65 70 75 80
Ala Gly Cys Gly Gly Cys Gly Thr Thr Gly Thr Thr Ala Gly Cys Cys
85 90 95
Gly Ala Ala Gly Ala Thr Gly Gly Gly Ala Cys Thr Ala Thr Cys Thr
100 105 110
Ala Thr Cys Ala Cys Gly Gly Cys Thr Gly Thr Ala Ala Thr Ala Thr
115 120 125
Thr Gly Ala Ala Ala Ala Cys Thr Cys Cys Gly Cys Thr Thr Ala Thr
130 135 140
Ala Gly Cys Ala Thr Gly Ala Cys Cys Ala Ala Thr Thr Gly Thr Gly
145 150 155 160
Cys Ala Gly Ala Ala Cys Gly Ala Ala Cys Ala Gly Cys Ala Thr Thr
165 170 175
Thr Thr Thr Thr Ala Ala Ala Gly Cys Thr Gly Thr Thr Thr Cys Cys
180 185 190
Gly Ala Cys Gly Gly Cys Gly Thr Ala Cys Gly Thr Ala Gly Cys Thr
195 200 205
Thr Cys Ala Ala Ala Gly Cys Gly Cys Thr Thr Gly Cys Ala Gly Thr
210 215 220
Ala Gly Thr Thr Gly Cys Ala Gly Ala Thr Ala Cys Ala Gly Ala Ala
225 230 235 240
Gly Gly Ala Cys Cys Thr Gly Thr Thr Thr Cys Cys Cys Cys Gly Thr
245 250 255
Gly Thr Gly Gly Ala Gly Cys Ala Thr Gly Cys Ala Gly Ala Cys Ala
260 265 270
Ala Gly Thr Ala Ala Thr Thr Gly Cys Gly Gly Ala Gly Thr Thr Thr
275 280 285
Thr Gly Thr Ala Ala Thr Gly Gly Cys Thr Cys Gly Ala Thr Gly Cys
290 295 300
Cys Thr Gly Thr Cys Thr Ala Cys Cys Thr Cys Ala Cys Gly Ala Ala
305 310 315 320
Cys Cys Thr Ala Ala Ala Ala Gly Gly Gly Gly Ala Cys Ala Thr Thr
325 330 335
Gly Ala Ala Gly Ala Ala Ala Cys Ala Ala Cys Thr Gly Thr Ala Gly
340 345 350
Cys Ala Ala Ala Ala Thr Thr Ala Thr Thr Ala Cys Cys Ala Gly Gly
355 360 365
Cys Gly Cys Ala Thr Thr Thr Thr Cys Ala Ala Ala Gly Gly Ala Gly
370 375 380
Gly Ala Thr Cys Thr Thr Thr Cys Ala Thr Ala Thr Gly Cys Ala Gly
385 390 395 400
Cys Ala Gly Ala Ala Cys Ala Ala Gly Gly Ala Gly Gly Ala Gly Gly
405 410 415
Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala
420 425 430
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly
435 440 445
Gly Ala Gly Gly Ala Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
450 455 460
Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
465 470 475 480
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Ala
485 490 495
Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
500 505 510
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
515 520 525
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
530 535 540
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
545 550 555 560
Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala
565 570 575
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
580 585 590
Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala
595 600 605
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
610 615 620
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
625 630 635 640
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
645 650 655
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
660 665 670
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
675 680 685
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly
690 695 700
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
705 710 715 720
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
725 730 735
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
740 745 750
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
755 760 765
Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
770 775 780
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala
785 790 795 800
Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
805 810 815
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
820 825 830
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
835 840 845
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr
850 855 860
Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys
865 870 875 880
Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys
885 890 895
Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys
900 905 910
Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala
915 920 925
Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr
930 935 940
Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly
945 950 955 960
Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys
965 970 975
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
980 985 990
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr
995 1000 1005
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
1010 1015 1020
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
1025 1030 1035 1040
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
1045 1050 1055
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
1060 1065 1070
Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
1075 1080 1085
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
1090 1095 1100
Ala Ala Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
1105 1110 1115 1120
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
1125 1130 1135
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
1140 1145 1150
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys
1155 1160 1165
Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala
1170 1175 1180
Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys
1185 1190 1195 1200
Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly
1205 1210 1215
Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala
1220 1225 1230
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly
1235 1240 1245
Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys
1250 1255 1260
Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys
1265 1270 1275 1280
Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly
1285 1290 1295
Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly
1300 1305 1310
Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys
1315 1320 1325
Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly
1330 1335 1340
Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys
1345 1350 1355 1360
Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly
1365 1370 1375
Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala
1380 1385 1390
Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala
1395 1400 1405
Gly Cys Ala Ala Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys Ala Ala
1410 1415 1420
Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly
1425 1430 1435 1440
Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys
1445 1450 1455
Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala
1460 1465 1470
Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly
1475 1480 1485
Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala
1490 1495 1500
Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr Gly Gly
1505 1510 1515 1520
Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys
1525 1530 1535
Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys
1540 1545 1550
Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly
1555 1560 1565
Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly
1570 1575 1580
Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly
1585 1590 1595 1600
Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala
1605 1610 1615
Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala
1620 1625 1630
Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys
1635 1640 1645
Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr
1650 1655 1660
Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr
1665 1670 1675 1680
Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys
1685 1690 1695
Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys
1700 1705 1710
Cys Ala Gly Cys Ala Ala Cys Ala Thr Thr Gly Gly Cys Gly Gly Cys
1715 1720 1725
Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala
1730 1735 1740
Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr
1745 1750 1755 1760
Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly
1765 1770 1775
Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Cys
1780 1785 1790
Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr Gly Gly Thr Gly Gly Cys
1795 1800 1805
Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys Gly Gly Thr
1810 1815 1820
Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys Ala Ala Gly Cys Gly Cys
1825 1830 1835 1840
Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Cys Gly
1845 1850 1855
Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Gly Cys Thr Gly
1860 1865 1870
Thr Gly Cys Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Gly Cys Cys
1875 1880 1885
Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Cys Cys Ala Ala Gly Thr
1890 1895 1900
Gly Gly Thr Gly Gly Cys Thr Ala Thr Cys Gly Cys Cys Ala Gly Cys
1905 1910 1915 1920
Ala Ala Cys Gly Gly Thr Gly Gly Cys Gly Gly Cys Ala Ala Gly Cys
1925 1930 1935
Ala Ala Gly Cys Gly Cys Thr Cys Gly Ala Ala Ala Cys Gly Gly Thr
1940 1945 1950
Gly Cys Ala Gly Cys Gly Gly Cys Thr Gly Thr Thr Gly Cys Cys Gly
1955 1960 1965
Gly Thr Gly Cys Thr Gly Thr Gly Cys Cys Ala Gly Gly Ala Cys Thr
1970 1975 1980
Ala Ala
1985

Claims (1)

1. An amplification method for analyzing the number of CGG repeats in an upstream untranslated region of an FMR1 gene, comprising: comprises the following steps:
(1) constructing a compound of three transcription activator-like effectors and deaminase which are covalently coupled, wherein the transcription activator-like effectors and the deaminase are flexibly connected by adopting 15 glycines, and the three compounds are CGG-TALE-downstream deaminase, TAA-TALE-downstream deaminase and CGG-TALE-upstream deaminase respectively;
(2) expressing the three complexes in escherichia coli;
(3) testing the activity of the protein expressed by the three compounds, mixing the three compounds after the determination to obtain the recombinant TALE-deaminase, and adjusting the activity of each compound to 1U/mu L;
(4) extracting target DNA, and adjusting the concentration of the target DNA to be between 100 and 500 ng/. mu.L;
(5) adding 10 mu L of reaction buffer solution and 1 mu L of recombinant TALE-deaminase into 10 mu L of target DNA, reacting for 4 hours AT 37 ℃, deaminating GC base in a specific region in the target DNA in a directional manner to convert the GC base into AT base, and inactivating for 10 minutes AT 95 ℃ after the reaction is finished;
(6) amplifying DNA after deamination reaction by PCR;
(7) determining the sequence of the PCR amplification product by adopting a Sanger sequencing method;
(8) and (6) analyzing the result.
CN202011096517.7A 2020-10-14 2020-10-14 Amplification method for analyzing CGG repeat number of upstream untranslated region of FMR1 gene Pending CN112176046A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866176A (en) * 2017-07-11 2018-11-23 北京阅微基因技术有限公司 The detection architecture and detection kit of a kind of pair of FMR1 gene 5 ' non-translational region CGG unit number of iterations
CN109355376A (en) * 2018-12-04 2019-02-19 深圳会众生物技术有限公司 Fragile X mental retardation FMR1 genetic test primer, kit and detection method
CN110577987A (en) * 2019-06-24 2019-12-17 胜亚生物科技(厦门)有限公司 Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866176A (en) * 2017-07-11 2018-11-23 北京阅微基因技术有限公司 The detection architecture and detection kit of a kind of pair of FMR1 gene 5 ' non-translational region CGG unit number of iterations
CN109355376A (en) * 2018-12-04 2019-02-19 深圳会众生物技术有限公司 Fragile X mental retardation FMR1 genetic test primer, kit and detection method
CN110577987A (en) * 2019-06-24 2019-12-17 胜亚生物科技(厦门)有限公司 Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof

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