mab unit 3 genetic engineering

Gene Cloning

Gene Cloning

Creating a copy of an organism or molecule from another entity

Restriction Enzymes

DNA-cutting enzymes that cleave phosphodiester bonds in DNA strands

Plasmid DNA

Circular, self-replicating DNA found in bacteria, used to carry and clone other DNA fragments

Recombinant DNA Technology

Technology that allows for the cutting and joining of DNA fragments, and insertion into a plasmid

Bacteriophage vectors

clone DNA fragments up to 15 kb; infect the bacterial species

Restriction Site

Specific sequence of bases in DNA recognized and cut by restriction enzymes

Palindrome

Sequence that reads the same forward and backward on opposite strands of DNA

Sticky Ends

Overhanging single-stranded ends of DNA fragments created by certain restriction enzymes

Blunt Ends

Double-stranded ends of DNA fragments created by certain restriction enzymes

Recombinant DNA

DNA formed by joining together DNA fragments from different sources

Transformation

Process of inserting foreign DNA into bacterial cells

Antibiotic Selection

Process of selecting transformed bacteria by growing them on plates with different antibiotics

Blue-White Selection

used to distinguish cells containing recombinant vs non-recombinant plasmids, utilizes lacz gene, and XGAL in agar

Dna Libraries

Collections of cloned DNA fragments from a particular organism stored i

Genomic DNA Libraries

Libraries containing fragments of an organism's entire genome

cDNA Libraries

Libraries containing complementary DNA synthesized from mRNA

Polymerase Chain Reaction (PCR)

Technique for amplifying specific DNA sequences in a short period of time

Taq DNA Polymerase

DNA polymerase isolated from Thermus aquaticus, used in PCR due to its heat stability

Cloning PCR Products

Rapid and effective method of cloning DNA fragments amplified by PCR

PCR primers

Primers are created to match the beginning and end sequence of certain sections

These are short DNA sections that set the boundaries for what will be copied

DNA probes are

short, single-stranded segments of DNA used in DNA hybridization experiments.- complementary to sequence of interest

multiple cloning site (MCS)

site that can be recognized by multiple restriction enzymes

cloning vector

DNA molecules that can carry foreign DNA into a host cell and replicate there.

gel electrophoresis process

enzymes are used to chop DNA from sample into fragments, fragments are then placed into wells; electrical current is run through the gel and fragments are attracted depending on charge; smallest/lightest/most charged move far, thus creating banding pattern used to identify and compare

DNA ligase

enzyme that chemically links DNA fragments together

bacterial artificial chromosome (BAC)

A large plasmid that acts as a bacterial chromosome and can carry inserts of 100,000 to 300,000 base pairs., cannot properly fold eukaryotic proteins

reverse transcriptase

An enzyme encoded by certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.. Used for cDNA libraries,

insulin

first protein made through recombinant DNA technology

Hybridization

binding of complementary DNA strands through hydrogen bonding. Used in library screening, DNA probes, Southern blotting, PCR

Linker sequences

synthetic DNA element in a cloning vector with unique restriction sites used for insertion of foreign DNA

Luciferase

Enzyme used in bioluminescence

Chemiluminescence

the emission of light from a chemical reaction

ethinium bromide

dye that binds to DNA in agarose gel, and fluoresces under UV light

CRISPR/Cas9

a unique technology that researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence, naturally occurring in bacteria

Southern Blot

A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting ds labeled piece of DNA is visualized when the filter is exposed to film.

Northern Blot

Similar technique [to Southern], except that Northern blotting involves radioactive DNA probe binding to sample RNA .

Western blot

Similar technique (to Southern ) except it involves antibodies binding to proteins

prodigiosin

The red pigment produced by S. marcescens.

dideoxynucleotide

type of nucleotide used during DNA sequencing to terminate synthesis

Sanger sequencing method

There are 4 test tubes each one containing ddNTP (only one), dNTP (all 4), DNA Polymerase, Primer, ssDNA template, . Synthesis occurs until a ddNTP is incorporated. You use gel electrophoresis to see the fragements. Read the gel bottom to top for the sequence.. Also known as chain termination sequencing, used in Human Genome project

FISH

Technique used to identify the location of a gene directly in a chromosome

RNAi (RNA interference)

inhibition of gene expression by RNA molecules, naturally occurring in bacteria

RT-PCR

A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction.

DNA microarray

A microarray of immobilized single-stranded DNA fragments of known nucleotide sequence that is used especially in the identification and sequencing of DNA samples and in the analysis of gene expression (as in a cell or tissue).

SOLiD Sequencing (NextGen)

sequencing technique uses short fragments of DNA (oligonucleotides) attached to beads

3rd generation sequencing

uses nanotechnology to cleave and sequence single nucleotides at a time

tryp operon

repressible operon; activated when tryptophan is absent, inactivated when tryptophan is present.