Download Concept 20.1 A. -Plasmid is the cloning vector.

Concept 20.1
A.
-Plasmid is the cloning vector.
- Expression of a Eukaryotic gene in a Prokaryote may be difficult because
of
a) Different aspects of gene expression:
- To overcome difficulties in promoters, and other control sequences we use
an expression vector.
- This vector contains a very active prokaryotic promoter just upstream of a
restriction site where eukaryotic gene can be inserted in the correct
reading frame.
- The bacterial will recognize the promotor and express the foreign gene.
b) Presence of introns (non-coding regions), in most Eukaryotic genes. These
make it hard to correct expression of the gene by bacteria, as they do not
have RNA splicing machinery.
- Use a cDNA form of the gene which only includes the exons of the gene.
-Bacteria can express a eukaryotic cDNA gene if the vector has a bacterial
promoter and control elements necessary for transcription.
-mRNA is used to make single stranded transcripts of DNA using reverse
transcriptase in vitro. The mRNA is then degraded and a second DNA strand
is made by DNA polymerase. This ds DNA is complementary DNA ( cDNA).
To overcome eukaryote-prokaryote incompatability scientists use eukaryotic
cells such as yeast.
- They are easy to grow and have plasmids.
- Also combined yeast artificial chromosomes (YACs) which have a eukaryote
origin of replication, a centromere, two telomeres with foreign DNA. Can
carry longer pieces of DNA, an entire gene rather than a fragment.
Many eukaryotic proteins need modification after translation ex. addition of
carbohydrate group etc.
So, need to get recombinant DNA into eukaryotic cells via electroporation.
H/W 24: PCR fig. 20.7 and animation site.
SNPs ( single nucleotide polymorphisms)Most of the diversity in these single base pair give rise to variations in
genome.
Detected by sequencing.
Occur about 1 in 1,000 base pairs.
You are 99.9 % identical to the person of the same gender!!!
Several million SNP sites identified.
Useful in studying more subtle effects of disease genes and evolution of
Man.
Gel electrophoresis allows separation of different sizes of DNA fragments.
Applications: DNA fingerprinting, isolating a cloned fragment.
Southern Blotting is a way to transfer DNA fragments onto a membrane for
probing to identify the fragment(s) of interest.
Applications: DNA fingerprinting, identifying specific fragment(s).
A probe is a short single stranded complementary sequence of DNA to
sections in the DNA fragment(s) being looked at.
PCR is a way to clone or make more copies of a segment of DNA you are
interested in studying.
Applications: forensics where you need enough DNA to analyze, want enough
DNA to sequence it, study it, mutate it, clone it.
Microarrays: If you want to see which genes are turned on when cells
become cancerous, or you take a certain medicine, then make cDNA for a
number of mRNAs and all of them screened using a labeled probe with a
complementary sequence to the gene being studied that has a fluorescent
tag.
Applications: Gene expression in a cancer cell compared to a normal cell,
looking at reaction to medications, screening for disease genes etc.