Molecular Techniques - Lecture 13

Question 1

What are DNA/Genomic libraries and how is the genomic data packaged? some examples of these libraries

Answer 1

Libraries are arrangement of large scale DNA/genomic sequences into fragments which are arranged in vectors such as plasmids or phage. Examples: cDNA, expression libraries

Question 2

Briefly describe the process involved in creating a DNA library

Answer 2

Large scale digest of vectors with REs»>Large scale digests of genes/DNA by REs (creating many fragments)&raquo_space;>ligate vectors with DNA fragments&raquo_space;>each fragment is integrated into one vector
Thus, one vector = has one unique DNA sequence = library

Question 3

Briefly describe the process involved in creating a genomic library

Answer 3

Large scale digest of vectors with REs»>Large scale digests of whole genome into many fragments by REs&raquo_space;>ligate vectors with genomic fragments»>each fragment is integrated into one vector
Thus, one vector = has one unique DNA sequence = genomic library = bits and pieces of the whole genome

Question 4

Briefly describe the process involved in creating a cDNA library

Answer 4

Isolate/purify mRNA&raquo_space;> rt to complementary DNA&raquo_space;> Large scale digest of vectors with REs&raquo_space;>Large scale digests of cDNA into many fragments by REs&raquo_space;> ligate vectors with cDNA fragments&raquo_space;> each fragment is integrated into one vector
Thus, one vector = has one unique cDNA sequence

Question 5

How do expression libraries differ from other type of libraries?

Answer 5

in expression libraries, Vectors can be transcribed&raquo_space; translated; thus making it possible to isolate protein of interest

Question 6

What is the basic principle of Blotting and what is southern(S), northern(N) and western(W) used for?

Answer 6

1.Separate by size (electrophoresis)&raquo_space; 2. denature and transfer from gel to solid membrane (blot)&raquo_space; 3. Add DNA labelled probes (in S & N) or antibody (in W) directly onto the solid membrane and incubate with ‘molecule’ 4. Detection - In S&N (hybridization of fragments to complimentary labelled DNA probe on the solid membrane); In W, proteins bind to labeled/tagged antibodies&raquo_space;> 5. wash and analyze for markers
Southern blot: DNA
Northern blot: RNA
Western blot: Protein
(Tip: secondary antibody may be used for W Blot to amplify signal)
Tip: Only Southern involves a denaturing step after DNA is put on the solid surface ton allow probe hybridization

Question 7

How does northern blot differ from reverse northern (relate this to their use!)

Answer 7

Northern blot is used to detect specific RNA molecules, while reverse Northern blot is used to identify/measure multiple RNA molecules based on their interactions with known DNA probes.

Question 8

***What type of genes are used in reverse northern blot as a comparative control? what are these genes?

Answer 8

Housekeeping genes are genes assumed to be found in every cell at the same level.

Question 9

***why are house keeping genes essential in reverse northern blots? what happens if they are not part of the experiment?

Answer 9

the blotting membrane in northern is then hybridized with a mixture of labeled probes representing known genes or sequences. The presence of specific RNA molecules in the sample is inferred based on the hybridization pattern with the probes. Hence, when comparing samples, the housekeeping genes evidence consistency in the quality of RNA loading technique as a control measure.
Tip: use own words as concept is KEY

Question 10

Explain the principle and process of microarray for measuring RNA briefly

Answer 10

isolate RNA from samples&raquo_space;> reverse transcription of RNA into cDNA (modified nucleotides containing a reactive group are incorporated into the cDNA during rt, thus a labelled cDNA»> cDNA hybridizes to the probes on the microarray slides»>emits fluorescence&raquo_space;>computational scanning
*If gene is expressed in a particular sample, that sample should bind/show fluorescence in the microarray slides

Question 11

Explain the principle and process of RNAseq and single cell RNAseq and its application

Answer 11

The techniques allow for the quantification of gene expression.
RNAseq: isolate mRNA&raquo_space;> cDNA&raquo_space;> sequencing (like illumina)&raquo_space;> computational analysis (analysis of graph peaks denoting the level of gene expression.
scRNAseq: same process but involves isolation of the mRNA from a single cell