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electrophoresis
migration of suspended particles in an electric field
restriction enzymes
an endonuclease that recognizes specific short sequences in DNA and cleaves the DNA molecule at or near that site
cloning
the production of many copies of  a gene or specific DNA sequence
PCR
polymerase chain reaction

procedure involving multiple cycles of denaturation, hybridization to oligonucleotide primers, and polynucleotide synthesis that amplifies a particular segment of DNA
blotting
procedure in which biological molecules in gel matrix are transferred onto nitrocellulose paper for scientific analysis north (RNA), south (DNA), west(proteins)
ELISA
enzyme linked immunosorbent assay

evaluate presence of antigen/antibody

detect substances with antigenic properties

high sensitivitiy

don't need radioisotopes
ethidium bromide
used as a fluorescence tag to identify nucelic acid band in electrophoresis

mutagenic
endonuclease
enzyme that breaks the strands of DNA at internal positions. some are involved in recombination of DNA
palindromic site
segment of DNA in which base pair sequence reads the same on 5' and 3' strand directions. has a point of symmetry
recombinant DNA
DNA molecule constructed in vitro by joining all or parts of 2 different DNA molecules
plasmid vector
vectors made from plasmids

extrachromosomal, double stranded circular DNA
cosmid vector
hybrids between plasmids and phage lambda chromosomes

replicate like a plasmid in ecoli

in vitro packaging of lambda chromosomes, to transform cells efficiently
dideoxynucleotides
ddNTP- chain terminating DNA precursers (nucleosidetriphosphates) with a hydrogen (H) linked to the 3' carbon in place of the 3' -OH used in DNA sequencing rxns.
genome
a complete set of chromosomes inherited as a unit from one parent
genomics
the study of the structure and function of entire genomes
functional genomics
the study of genome function

analysis of transcriptome
proteome complete set of proteins
rna transcribed
comparative genomics
study of genetic evolution of species
evolution of cancer cells from early to late stages
SNP
single nucleotide polymorphism

a single base pair in teh DNA that varies in a population
haplotype
set of linked genetic variants especiall SNP on a chromosomes

complete set of DNA sequence for one individual

set of single nucleotide polymorphisms on a chromosome
expression profiling
tumor typing
screens all mRNA in tumor
make/label cDNA
hybridize to array
vector
agent used to carry genes into another organism
RFLP
restriction fragment length polymerization

DNA cut into small fragments via restriction enzymes

separated by electrophoreses and southern blotting

visualized by hybridizing membrane with lableled DNA probe

used to detect 2 or more genetic variants
DNA electrophoresis
molecules are suspended in a gel and an electric current is introduced with corresponding cathod and anodes. the molecules move to either locations by charge and size, with te higher charge/smallest move the furthest. Can use garase or polyacrylamide gel
How did restriction enzymes evolve?
Restriction enzymes evolved from a mechanism found in prokaryotes. The prokaryotes used this to protect its DNA from intruder (virus) DNA. the cells DNA was methylated for protection and the restriction enzymes degraded any DNA taht wasn't methylated, removing the viral DNA
RFLP uses
genetic mapping
genetic counseling
paternity tests
criminal identification
Cloning process
to clone you need to isolate gene of interest with restriction enzymes. then you restrict a vecor and mix it with your gene (bind with ligase). Introduce recombinant into a host cell and grow host.
PCR various steps
required:
free NTPS
buffer
Mg2+
thermal cycler
taq pol
template, primers

initial-
denature dsDNA at 94C 2 min
primer anneal at 50-60C 30 sec
elongation 68-72C 1.5 mins

2-30
denate dsDNA 94C 30 sec
anneal
elongate

cycle 3 is the first time dsDNA is seen as a product
PCR applications
  • creates dna fast/ only a few hourse
  • create hybridization probes
  • dna selective isolation
  • forensic analysis
  • disease diagnosis
  • pathogen subtypes
what is required for PCR?
  • template DNA
  • buffer
  • Mg2+
  • taq pol
  • thermal cycler
  • template & two primers
sanger sequencing
dideoxynucleotides are added and terminate when ddATP added. Will create lengths that end at all possible NT in chain. All have common 5' end. 1% ddATP in solution

four different rxns using different ddNTP (polyacrylamide on all 4) or all in one
gel is read anode to cathode
nascent 5'-3'
complementary 3'-5'
Real time PCR
allow for detection of PCR amplification during early phases of RxN

measures kinetics of rxn in early phases of PCR (traditional uses agarose and only see end product)
3 phases
exponential, linear, plateau where rxn is stopped
veiw increase in DNA as it is amplified
rapidly detect nucleic acids in diagnostics
blotting
transfer of electrophoretically separated polypeptides onto a solid support medium (nitrocellulose) for further analysis)
southern hybridization
detection of specific dna sequence in DNA

probe hybridzation for fragment detection

single DNA fragment with specific sequence whose presense in target DNA is to be determined

radioactivity or fluoresence for detection

probe washed then gel viewed
Significance of SNPs
occures when single NT differs between members of seme alleles

single nucleotide polymorphism
determine evolutionary history
more shared SNPS, more closely related
genetic markers for disease
genes predict individuals disease susceptibility
Microarrays benefits and drawbacks
contain alrge number of genes and small in size
thousands of hybridization probes
compare gene expression between tissues
mrna fluorsecently labled
oligo-short DNA more dense

drawback
lack fo standardization
variable outcome
cost
souther hybridization
process by which DAN that has been transferred onto a membrane is made single stranded then hybridized to a complementary single stranded probe
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