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PV92 Alu Insertion (Lab Report Sample)
I was given forward and reverse DNA primers. I was expected to BLAST it over the NCBI databases, then provide answers for given questions.source..
PV92 Alu Insertion
It is important to note that alignment sequence was done based on the instructions given in the laboratory manual. The chosen database was denoted as "Genome (all assemblies top - level)â€. This was the most preferred choice since "Genome (all assemblies top-level, Annotation Release 106)â€; was not there amongst given list of choices. Thus, results differ slightly from the answers given.
On which human chromosome did BLAST find a sequence alignment?
Answer: Chromosome 16
Homo sapiens Chromosome 16, GRCh38.p2 Primary Assembly; Sequence ID: ref|NC_000016.10| Length: 90338345, Number of Matches: 2. Hit score for the reverse primer was 49.1 bits (26), e-value was 2e-04, identities 26/26 which is equivalent to a 100% match with zero gaps introduced. The forward primer hit score was 47.3 bits (25) with an e-value of 9e-04, identities 25/25 which equivalents to a 100% match therefore, and no gaps were introduced.
Figure 1: http://blast.ncbi.nlm.nih.gov/Blast.cgi
What is the name of the gene with which the PV92 forward primer aligns (see all headed "featuresâ€)?
The name of the gene is Cadherin 13 isoform 2. The human chromosome number 16 has high segments of duplicate nucleotide sequences. Based on Martin, et al., (2004), the chromosome contains a total of 78,884,754 base pairs as reported in the completed Human Genome Project. Manual annotation demonstrates that there are a total number of 880 protein-coding sequences, which have been verified by 1,670 transcripts alignments, 19 RNA gene sequences. It also contains 341 pseudogenes as well as 3 RNA pseudogenes (Martin, et al., 2004).
One of the genes is the cadherin gene. It encodes for calcium-dependent as well as membrane associated glyco-proteins. The uniqueness of encoded protein is that it does not have the pro-sequence as well as the tripeptide HAV binding recognition sequences which are characteristic of many types of cadherins (Martin, et al., 2004).
How good is the alignment (how many nucleotides align with the 25-long primer)?
25/25 100%, it is clear that there was no gaps which were introduced during the alignment. Thus the 25-long primer completely matched the query sequence. More information is as highlighted in Figure 2 below.
Figure 2: http://blast.ncbi.nlm.nih.gov/Blast.cgi
What is the e-value, also called "expect," for this alignment?
2e-04 for the reverse primer and 9e-04 for the forward primer: Figure 2 above shows the query results for both.
What is the "Range" of the alignment (i.e., the numbered base-pairs, starting from one end of the chromosome) with which the forward primer sequence aligns)?
The answers given before the search are in contrary with what was determined later. The previous results suited that of Homo sapiens chromosome 16, alternate assembly CHM1_1.1 sequence, as shown in Figure 3 below (Martin, et al., 2004). A summary of alignment results is as follows;
A hit score of 47.3 bits (25), e-value of 9e-04, identification level at 25/25 (0%), no gaps, and range of between 84323157 to 84323181.
A hit score of 49.1 bits (26), e-value of 2e-04, identification level of 26/26 (100%), and no gaps. Its range was determined to lie between 84323563 to 843235588.
Figure 3: http://blast.ncbi.nlm.nih.gov/Blast.cgi
However, the sequence to be used for this report was specified as that from the Homo sapiens chromosome 16, GRCh38.p2 Primary Assembly, gi|568815582|ref|NC_000016.10|. The answer to this particular question will be based on this sequence. Figure 4 below shows the derived results.
The forward primer recorded a hit score of 47.3 bits (25) with the sequence in the database. It had an e-value of 9e-04, 100% identification value with no introduction of gaps when aligning. Its range was found to lie between 82878195 to 82878219. Length is 25.
The reverse primer recorded a hit score of 49.1 bits (26), an e-value of 2e-04 (100%) with no gaps introduced during the alignment process. The range for this sequence was determined to lie between 82878585 to 82878610. Length is 26.
Figure 4: http://blast.ncbi.nlm.nih.gov/Blast.cgi
How many base-pairs long is the PCR fragment that would be expected from use of these two primers?
The entire mapped sequences ranges from 82878195 to 82878610. As per the instructions given on the laboratory, the lowest range for the forward primer was 82878195 and the greatest range for the reverse primer was identified as 82878610. Please refer to Figure 4 as shown above. The number of base pairs which can be derived from both primers will be 82878610 minus 82878195, which is gives a total and DNA product of 415 base pairs in length.
What tissue is this gene associated with?
Cadherins are proteins which are overly expressed in the heart tissues as compared to other tissues such as the ...
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