First discovered in bacterium.

allow us to cut large DNA molecules into smaller and ore easily manipulated fragments .

recognize specific DNA sequences. cleave specific phosphodiester bonds within a spequence.

sequences cleaved are palindromic (words that can read backward) producing sticky ends.

a carrier that carries the desired DNA to be cloned. vector is usually combined with a plasmid (naturally occuring bacteria's circular DNAs).  The recombinanat plasmids are introduced into bacteria. as the bacteria grow, the plasmids replicate and large nuber of identical copies are made.

plasmid holds up tp 10kb

bacteriophage holds up to 25 kb

collection of all DNA sequences in an organism's.

may contain intron and intergenic squences

the cDNA libary consists of double-strand DNA that is complementary to the cellular mRNA expressed in the cells/tissues from which it is obtained.

The cDNAs in such a library are heterogeneous, each representing the genes transcribed into mRNA at the time of isolation of the mRNA for the library.

What are the requirements for PCR?

requirement:

DNA template

Defined Oligoprimer that flank the target sequence

Heat-Stable DNA polymerase

dNTPs

1. Denaturation of the double stranded DNA at high temp

2. Annealing of oligo primer to the DNA template at low tem

3.Extension of primers using DNA polymerase (taq-thermal aquaticus)

typically repeat 25-50 cycles

-finding DNA seuqences/genes in a libary can be done using DNA hybridization techniques.

Probes: oligo probe synthesized and tagged with fluorescent. DNA probes base-pair with known complementary sequences. can be used to screen a gene libary.

1. DNA is cut with restriction enzyme into smaller DNA pieces called restriction fragments

2. restrcition fragments are separated by size on agarose gel

3. a replica of the DNA in gel is transfer onto a membrane filer by blotting. this is done by capillary action.

4. DNA is denatured to single strands by alkaline solution and is transferred from the gel to a strong mem such as nitrocellulose.

5. Hybridization: specific probes are hybrisized to the DNA on the filer.

6. complementary DNA sequences bound to labeled probes are visualized.

STRs. typically 4-5 nucleotide repeat present in non-coding region.

are excellent genetic markers due to high level of polymorphism.

STRs of each person vary widely, unique patterns.

1. amplify DNA by PCR

2. Primers that flanking the locus are used and two bands (maternal and paternal) for a locus will be seen on gel.

3. STR analysis looking at multiple STR loci.

CODIS (combined DNA index system) is a DNA data base funded by the United States. It is a computer system that stores DNA profiles created by federal, state, and local crime laboratories in the United States.

advanced hybridization technique

allows the expression of thousands of transcripts to be analyzed at the same times. can be detected and quantified at the sametime.

1. DNA microarray chip is spotted with large number of DNA sequences at defined location.

2. mRNAs obtained from two different cell population are converted to cDNAs that are labeled in different colors using different fluorescent dyes.

3. the samples are mixed together and allowed to hybridize to the DNA fragments on the DNA microarray.

4.Washing away the microarray to remove unbound molecules.

5. scan the chip to show the intensity of the fluorescence at each probe spot. this intenstity is proportional to the number of copies of that specific mRNA in the original mRNA population. 

The intensity of that same spot in both channels is compared. this result in a measurement of the ratio of transcript level for each gene being studied.

Cancer research-identification of single gene products that are expressed in cancer cells but not in normal tissue.

possibly to classify many cancers based on thier gene expression profiles.

RNA interference (RNAi)

miRNA (microRNAS) small non-coding RNAs. they regulate gene expression in cell.

act as silencer.

1. miRNA with a sequence complementary to the target mRNA that is to be TURNed Off is put nto a cell.this miRNA becomes part of a protein assembly called a RISC (RNA-induced silencing complex where it is cut into single strands called siRNAS.

2.mRNAS in the cytoplasm with a sequence complementary to the siRNA bind to it. Translation of the mRNA is blocked., and the mRNA is degraded.

could be used to silence ocogenes.