L10: Constructing Transcript Maps

Question 1

Hybridization to RNA/cDNA

Answer 1

A candidate DNA clone can be hybridized against a northern blot containing a panel of mRNA isolated from different tissues. Positive signal indicates the presence of a gene within the cloned fragment.

Question 2

Zoo blot hybridization

Answer 2

Coding regions are conserved. Noncoding regions are not well conserved among species.
o Observe an allele in different species

Question 3

CpG island identification

Answer 3

CpG islands are short (1 kb), hypomethylated GC-rich sequences which are often found at the 5’ ends of vertebrate genes. 56% of genes are associated with such sequences.

Question 4

Exon trapping identifies expressed sequences

Answer 4

Exons can be identified in cloned genomic DNA by subcloning the DNA into a suitable expression vector (pSPL3) and transfecting into an appropriate eukaryotic cell line. RT-PCR using primers within vector exon sequences to retrieve the trapped novel exons.

Question 5

Transcripts maps

Answer 5

RNA-seq data or Individual EST (expressed sequence tag) clones are mapped in-silico or by PCR to a chromosome, a subchromosome or to defined genomic clones.

Question 6

Computer analysis

Answer 6

homology searches and exon prediction programs

Question 7

Expression can be measured at RNA or protein level

Answer 7

RNA
Tissue/cellular in situ hybridization =high resolution
DNA microarray hybridization/SAGE=high throughput

Protein
Immunocytochemistry/Immunofluorescence=high resolution
Gel electrophoresis/mass spectrometry=high throughput