Lecture 10 - Genetic Engineering and Recombinant DNA Flashcards
No product or application is directly derived from it
basic science
Applications from basic research science
applied science
- Clip DNA crosswise at selected positions
- Recognize foreign DNA
- Capable of breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA
- Protect bacteria and archaea from bacteriophage or plasmids
restriction endonuclease
- Each endonuclease recognizes a sequence of 4 – 10 base pairs
- Recognize and clip at palindromes
restriction endonucleases
Staggered symmetrical cuts that leave short tails
sticky ends
Pieces of DNA produced by restriction endonucleases
restriction fragments
- Differences in the cutting pattern of specific restriction endonucleases give rise to restriction patterns of different lengths
- Allows direct comparison of DNA of two different organisms at a specific site
Restriction fragment length polymorphisms (RFLPs)
- Necessary to seal sticky ends together by rejoining the phosphate-sugar bonds cut by endonucleases
- Main application is final splicing of genes into plasmids and chromosomes
ligase
Replicates HIV and other retroviruses
Able to convert RNA into DNA
reverse transcriptase
- Made from messenger, transfer, ribosomal, and other forms of RNA
- Useful in synthesizing eukaryotic genes from mRNA transcripts
- Gene is free of introns
complementary DNA
Positions of DNA fragments are determined by staining the DNA fragments in the gel
gel electrophoresis
Useful in characterizing DNA fragments
Allow for comparison of genetic similarities among samples in a genetic fingerprint
electrophoresis
Two different nucleic acids can ____ by uniting at their complementary regions.
hybridize
ssDNA can hybridize with
ssDNA or RNA
RNA can hybridize with
other RNA
A short stretch of DNA of a known sequence that will base-pair with a stretch of DNA with a complementary stretch of DNA if it exists in a sample
gene probes
carry reporter molecules such as fluorescent dyes so that areas of hybridization can be visualized
gene probes
detected when non pigmented substrates become color molecules by the action of the enzyme
enzyme linked probes
Diagnosing the cause of an infection from a patient’s specimen
Identifying a culture of an unknown bacterium or virus
gene probes use
DNA is isolated, denatured, placed on an absorbent filter, and combined with a microbe-specific probe
Blot is then developed and observed for areas of hybridization
hybridization
Probes are applied to intact cells
Observed microscopically for the presence and location of specific genetic marker sequences on genes
FISH
A very effective way to identify genes on chromosomes
Also effective in identifying bacteria living in natural habitats without culturing them
Used to detect RNA in cells and tissues
FISH
Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques.
PCR
Can detect cancer from a single cell
Can diagnose an infection from a single gene copy
PCR
uses the same events of DNA replication
PCR
synthetic oligonucleotides of a known sequence of 15 – 30 bases that indicate where amplification should begin
primers of PCR
High temperatures necessitate use of DNA polymerases isolated from thermophilic bacteria
DNA polymerase of PCR
3 steps of PCR
denature
priming
extension
two good types of cloning vectors
plasmids
bacteriophages
- small, characterized, easy to manipulate
- can be transferred via transformation
- carry genetic markers for resistance to antibiotics
plasmid vector
natural ability to inject DNA into bacterial hosts through transduction
bacteriophageRecombinant organisms produced through the introduction of foreign genes
Also known as genetically modified organisms (GMOs)
Recombinant organisms produced through the introduction of foreign genes
(GMOs)
transgenic organisms
creation of organisms from scratch
synthetic biology
a result of synthetic biology
XNA
- Contain different sugars than DNA and RNA, or in some cases, no sugars at all
- Enzymes were able to copy resulting in the ability to store information and pass it on to descendant cells.
- These findings imply that life on other planets does/did not require DNA or RNA to exist
XNA
give an exact order of bases in a plasmid, chromosome, or genome
sequence maps
The whole genome of an organism is broken down into smaller, manageable fragments
Fragments are separated through gel electrophoresis (inserted into plasmid and cloned into an E coli cell)
whole genome shotgun sequencing WGSS
Plasmids are purified and DNA fragments are sequenced by automated sequencers
A computer program takes all the sequence data and is able to find where the sequence overlaps
WGSS
a larger, contiguous set of nucleotide sequences produced by the automated process
contigs
Sophisticated computers and systems developed to sequence genomes in a shorter period of time with less human effort
high-throughput sequencing
Manage the data produced through high-throughput screening
Analyze and classify genes
Determine protein sequences
Ultimately determine the function of the genes
genomics and bioinformatics
Determining the functional information of a gene sequence
annotating the genome
Based on the principle that the way nucleotides are combined is different for each organism
dna profiling
only a single nucleotide is altered
SNP
Allows biologists to view the expression of genes in any given cell
Track the expression of thousands of genes at once
microarray analysis
study of the proteins a cell produces at a defined point in time
proteomics
