Lecture 14: Genetics Technologies Flashcards
how are restriction enzymes produced?
by bacteria as a defense mechanism against infection by viruses
how many restriction enzymes have been identified?
- 3500
- 150 are commonly used
recognition sequence
a specific nucleotide sequence recognized by restriction enzymes, cut by endonuclease activity
what are the purposes of restriction enzymes?
- making recombinant DNA and appraising success for research, medicine, and agriculture
- DNA profile anlysis for disease diagnosis, paternity/family relationship testing, and forensics
what are the protruding ends that were cut by restriction enzymes called?
sticky ends
what is the purpose of restriction fragments?
making recombinant DNA
how is recombinant DNA made?
when you cut two separate molecules of DNA with the same restriction enzyme, the fragments will have matching sticky ends
RFLPs
- restriction fragment length polymorphism
- marker
- presence of absence of restriction sites to be digested by restriction enzymes
VNTR
- variable number of tandem repeats
- marker
- in the human genome, there exists short repetitive portions of DNA. the lengths of these repetitive sequences will vary from individual to individual, producing unique banding pattern
SNPs
- single nucleotides polymorphisms
- marker
- rare substitution differences, about .001 frequency in humans (1 SNP every 1000bp). SNPs often constitute different alleles
PCR sites
- marker
- presence or absence of priming sites, the ability for primers to anneal to an individual’s DNA
what are the steps for RFLP?
- DNA extraction
- PCR with labeled primers
- digestion with restriction enzymes
- electrophoresis separation
- laser detection
vectors
carrier DNA molecules that transfer and help replicate inserted DNA fragments
how do vectors vary?
- differ in the hosts they are able to enter
- size of inserts they can carry
what should vectors include?
- should contain several restriction enzyme cleavage sites that allow insertion of DNA fragments to be clones
- they must be able to independently replicate themselves and any DNA fragment they carry once they are inside a host cell
selectable marker genes
to distinguish host cells that have taken up vectors from those have not, usually an antibiotic resistance gene or a color changing gene
plasmid
- vector
- 10-20 kb of target DNA
bacteriophage
- vector
- lambda can carry a 15kb insert
cosmids
- vector
- a plasmid + lambda DNA hybrid, 50kb inserts
bacterial artificial chromosomes (BACs)
- vector
- F plasmids that can accept 150-300kb inserts
yeast artificial chromosomes (YACs)
- vector
- can accept big inserts, most useful in eukaryotes (~1M)
polymerase chain reaction (PCR)
- rapid method of DNA cloning used to generate millions of copies in short time
- copies specific DNA sequence via in vitro reactions; can amplify target DNA sequences present in very small quantities
what are the components of PCR?
- two primers - both primers anneal to denatured DNA
- dsDNA to be cloned is put in a tube with DNA polymerase, Mg 2+ and dNTPs
- complementary strands are synthesized by heat-stable DNA polymerase
what are the three steps of PCR?
- denaturation
- primer annealing
- extention
- steps are repeated over and over using a thermocycler to amplify DNA exponentially
what are the limitations of PCR?
- need for target DNA sequence information
- minor contamination from other sources can cause problems
- short size and limiting amounts
- primers have self-annealing regions within each primer preventing its annealing to target DNA
reverse transcription PCR (RT-PCR)
- methodology for studying gene expression
- reverse transcriptase is used to generate ds-cDNA
quantitative real-time PCR (qPCR)
-real time PCR allows researchers to quantify amplification reactions as they occur in real time
DNA ligase
- DNA fragments will seal phosphodiester backbone
- joints restriction fragments covalently to produce intact DNA molecules
multiple cloning sites
- restriction sites for commonly used restriction enzymes
- allow scientists to clone a range of different fragments
what are the applications of PCR?
- most widely used technique in genetics and molecular biology
- allows for screening of mutations involved in genetic disorders
- location and nature of mutation can be determined quickly
reverse transcription PCR (RT-PCR)
- methodology for studying gene expression (mRNA production by cells or tissues)
- reverse transcriptase is used to generate ds-cDNA
quantitative real-time PCR (qPCR)
-real time PCR allows researchers to quantify amplification reactions as they occur in real time
vectors
- carrier DNA molecules
- can replicate cloned DNA fragments in host cell
- must be able to replicate independently
- have several restriction enzyme sites to allow insertion of DNA fragments using ligase
- carry selectable gene marker to distinguish host cells that have taken them up from those that have not
plasmid
- extrachromosomal double-stranded DNA molecule
- replicates independently from chromosomes within bacterial cells
plasmids used in DNA cloning
- genetically modified bacterial plasmids - first vectors developed
- engineered to contain: a number of convenient restriction sites and a marker gene to select for presence in host cell
DNA ligase
- DNA fragments will seal phosphodiester backbone
- joins restriction fragments covalently to produce intact DNA molecules
transformation
- plasmids are introduced into bacteria via transformation
- two main techniques: using calcium ions and brief heat shock to pulse DNA into cells and electroporation
electroporation
a brief but high intensity pulse of electricity to move DNA into bacterial cells
