A technique of amplification that results in clones of identical cells containing the recombinant DNA molecule.

Donor DNA is fragmented my restriction enzymes and inserted into a vector.  The donor DNA and vector DNA together are called recombinant DNA.  The cell replicates and results in a clone of the recombinant.

Restriction enzymes "cut" the DNA

DNA Ligase "glues" the DNA

A sequence of letters that can be read the same forwards and backwards.

*RACECAR*

5' GAATTC 3'

3' CTTAAG 5'

Plasmids, modified bacterial viruses, Cosmids, PAC, BAC and YAC

Plasmid Vectors:  replicate independent of bacterial chromosome.  Routinely used are those that carry genes for drug resistance.

Bacteriophage Vectors:  Ask Dr. G to Explain

Cosmids:  Vector for larger DNA inserts.  Can carry 35-45kb inserts.  Engineered hybrids of lambda phage particles which act as syringes. 

YAC (yeast artificial chromosome):  For insterts larger than 300kb.

What is a genomic library?

 A genomic library is a collection of recombinant DNA in either bacterial or phage chromosomes that together make up the entire genome of the donor DNA.

A molecule (either DNA, RNA or protein) that recognizes and marks the desired clone. 

This is a screening technique screen for the fragment of interest.

Those that recognize a specific nucleuc acid sequence

Those that recognize a specific protein

Gel electrophoresis separates the molecules in the mixture by size. 

A mixture of linear DNA molecules is placed into a well in an agarose gel.  The well side of the gel is attached to the cathode (+).  The molecules contain charges themselves and move towards the opposite end of the gel towards the anode (-).  The speed of diffusion through the gel is inversely dependent on their size.  Bands can be visualized by staining with ethiduim bromide which fluoresces under UV light.  If the fragments are separated well enough, the fragments can be cut from the gel and the DNA sample can be purified.  Therefore, DNA electrophoresis can be either diagnostic or preparative.

PCR= Polymerase Chain Reaction

1. Start with a solution containing DNA source, the primers, deoxyribonucleotides (AGCT), and a heat tolerent DNA polymerase (Taq polymerase).

2. Denature DNA

3. Primers hybridize to their complementary sequences in the ssDNA molecule in a cooled solution.

4. Taq Polymerase extends the DNA segment from the primers.

5. Complementary new strands are synthesized forming two double stranded DNA molecules identical with the parental dsDNA molecule

**This represents one cycle**

Three cycles are required to completely isolate a fragment of interest.  After about 25 cycles, the target sequence has been amplified about 10^6 fold.

Most popular method:  Sanger Sequencing

A cloned DNA segment can be sequenced by characterizing a serial set of turncated synthetic DNA fragments, each terminated at different positions corresponding to the incorporation of a dideoxynucleotide.

Amniocentesis is a procedure that allows fetal cells to be taken from the anmiotic fluid and analyzed to identify a number of known disorders.

Chronic Villus Sampling is a related procedure in which a small sample of cells from the placenta are aspirated out with a long syringe.  CVS can be performed earlier in pregnancy.

Recombination maps:  map the loci of genes that have been identified by mutant phenotypes showing single-gene inheritance.  Positions are not relative distances and have to do with linkage.

Physical maps:  show the genes as segments arranged along the DNA molecule that constitutes a chromosome.

By determining the frequency of recombinants

Measured in map units (mu)

total recominants/total progeny=%recombinants=mu

What can affect distances in recombination maps?

(Why are they less accurate than distance maps?)

Multiple Crossovers (Distance)

Location of gene "hot spots"

Other Biological factors (Age, Sex, Genotype, Environmental Influences)

They are less accurate because we fail to take into account the true # of recombinants.

Haldane function

Kosamm function

Perkins fucntion (for fungi)

These are mathematical functions used to get around the multiple crossover problem.

What are the common phenotypic ratios expected from testcrosses?

Mono, Di, Trihybrid?

Monohybrid 1:1

Dihybrid 1:1:1:1

Trihybrid 1:1:1:1:1:1:1:1