Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferable using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.

The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferable using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.

Methods of detecting DNA sequences from multiple pools comprising at least one species of DNA molecule comprise combining the pools to form a combination pool; in the combination pool, generating at least one linear DNA concatemer containing one DNA molecule from each pool, wherein a position of each DNA molecule within the concatemer correlates to the pool from which the DNA molecule originated; and sequencing the concatemers, thereby detecting the DNA sequence of each DNA molecule at each position in each concatemer, wherein each detected DNA sequence is assigned to the pool from which its DNA molecule originated based upon its position within the concatemer.

Methods of detecting DNA sequences from multiple pools comprising at least one species of DNA molecule comprise combining the pools to form a combination pool; in the combination pool, generating at least one linear DNA concatemer containing one DNA molecule from each pool, wherein a position of each DNA molecule within the concatemer correlates to the pool from which the DNA molecule originated; and sequencing the concatemers, thereby detecting the DNA sequence of each DNA molecule at each position in each concatemer, wherein each detected DNA sequence is assigned to the pool from which its DNA molecule originated based upon its position within the concatemer.

The invention relates to a method for detecting cell death using PCR (polymerase chain reaction) techniques, including qPCR/quantitative PCR or ddPCR/digital droplet PCR, or any other technique for detecting a small amount of DNA (such as nanostrings).

The invention relates to a method for detecting cell death using PCR (polymerase chain reaction) techniques, including qPCR/quantitative PCR or ddPCR/digital droplet PCR, or any other technique for detecting a small amount of DNA (such as nanostrings).

Methods for determining the presence or absence of expansion of CGG repeat sequence in the FMR1 gene presence or absence of expansion of CCG repeat sequence in the FMR2 gene are provided. The methods are useful in identifying an individual with normal/intermediate, versus premutation or full mutation allele of FMR1 gene and FMR2 gene due to the expansion of CGG repeats and CCG repeats in the 5′-untranslated region respectively. The methods are also useful for screening newborns for fragile X syndrome or for screening women to determine heterozygosity status with full premutation of the CCG repeat tract. The methods are also useful in estimating the premutation and full mutation carrier frequency and estimating the prevalence of FXTAS AND FXPOI in a population. The methods are simple, rapid and require small amount of sample.

Methods for determining the presence or absence of expansion of CGG repeat sequence in the FMR1 gene presence or absence of expansion of CCG repeat sequence in the FMR2 gene are provided. The methods are useful in identifying an individual with normal/intermediate, versus premutation or full mutation allele of FMR1 gene and FMR2 gene due to the expansion of CGG repeats and CCG repeats in the 5′-untranslated region respectively. The methods are also useful for screening newborns for fragile X syndrome or for screening women to determine heterozygosity status with full premutation of the CCG repeat tract. The methods are also useful in estimating the premutation and full mutation carrier frequency and estimating the prevalence of FXTAS AND FXPOI in a population. The methods are simple, rapid and require small amount of sample.

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.