Module 4: Molecular Cell Biology

Front 1

Cloning

Back 1

the process of creating several individuals from one progenitor that are genetically identical. (can be conducted with bacteria, viruses, or eukaryotes)

Front 2

Enzymes involved in cloning

Back 2

-Restriction enzymes act by cutting the DNA at specific sites and can reform base pairs to produce either the two original pieces or mixed pieces.
-Ligase serves to reseal the DNA by re-creating circular DNA.

Front 3

Library types

Back 3

-Genomic
-cDNA

Front 4

Genomic libraries

Back 4

-has inserts derived from all the DNA of a cell.
-DNA is isolated, cut with enzymes, and inserted into vectors.
-All DNA of nucleus present so all genes are present. All regions wanted will be present, but the piece of interest is very rare.
-Have to use multiple screens to find the clones you want. Also, a gene may not be contained within a single insert. If a restriction site comes in the middle of the gene, your gene will be found in two clones. Some clones will contain more than one gene.
-It is difficult to use genomic clones to determine the amino acid sequence of proteins since all the introns are present.

Front 5

cDNA libraries

Back 5

-made from active DNA in the cell.
-This DNA is isolated by isolating the mRNA of the cell and using reverse transcriptase to make DNA copies of the mRNA.
-The DNA of interest is present in amounts corresponding to its concentration in the mRNA, and thus highly transcribed genes will be highly represented among the clones.
-Each clone will contain only one gene, and since no introns are present in mRNA, the sequence of the proteins can be easily figured out.
-The disadvantage is that there is not always a complete copy of the RNA so regions of the gene (especially the beginning) can be missed. No knowledge of introns or regulatory sequences gained. -Must collect tissue specific to cell type expression of interest

Front 6

Screening techniques (Name the types: 7)

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-probe complementary to the DNA of interest
-probe from same/related gene in similar organism
-plus/minus screening
-Chromosome walking
-antibody isolation
-transposon tagging
-rescue screening

Front 7

Plus/minus screening

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you make two probes, one from mRNA that contains the mRNA of interest (the plus probe) and another from mRNA that does not (the minus probe). The probes are made by reverse transcriptase, labeled as before and hybridized to two membranes taken from the same plate.
-those that hybridize to plus probe are potential candidate

Front 8

Chromosome walking

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-1st probe=made from piece of DNA located on same chromosome as area of interest.
-2nd probe=made from regions next to area of interest on chromosome (found by 1st probe) and is used to find overlapping clones.
-In this way, the molecular biologist walks along the chromosome until the DNA of interest is found. Very slow and tedious process (that can be messed up easily, i.e. hybridizing more than one gene).

Front 9

Transposon tagging

Back 9

Mutations produced by these transpositions can be used as a cloning tool. This requires a genomic library made from the mutant individuals

Front 10

Rescue screening

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the cells to be transformed have mutations in the gene of interest and are transformed and plated on a medium that will allow non-mutants to be seen.