7.1 Flashcards
Molecular Biology
The study of the structure and functions of nucleic acids and proteins.
Recombinant DNA
A molecule of DNA composed of genetic material from different sources.
Restriction Enzymes
Enzymes that can cleave hydrogen bonds and cut DNA.
Restriction Endonuclease
An enzyme that cleaves (cuts) the interior of double-stranded DNA in a sequence-specific manner.
Target Sequence
A short sequence of DNA that is recognized by a restriction enzyme.
Restriction Site
The point on a DNA sequence where the strand is cut.
Sequence Specificity
The cuts made by restriction endonucleases are specific and predictable. The same enzyme will cut a particular strand of DNA the same way each time, producing an identical set of DNA fragments.
Restriction Fragments
A small segment of DNA generated by cutting a larger piece of DNA with a restriction enzyme.
Staggered Cuts
Most restriction endonucleases produce a staggered cut that leaves a few unpaired nucleotides on a single strand at each end of the restriction fragment. These are called sticky ends or overhangs.
Step One of Making Recombinant DNA
A restriction endonuclease is selected that can cut both DNA fragments to be combined.
Step Two of Making Recombinant DNA
Each piece of DNA is reacted with the restriction endonuclease enzyme to produce cut DNA fragments.
Step Three of Making Recombinant DNA
The two cut DNA fragments are incubated with another enzyme, DNA lipase. This enzyme seals the breaks in the DNA, forming covalent bonds between the 2 different fragments.
Gene Cloning
The process of manipulating DNA to produce many identical copies of a gene or another segment of DNA in foreign cells.
Plasmid
In recombinant DNA technology, a self-replicating, closed circular piece of DNA that can act as a carrier of a gene to be cloned in bacteria .
Step One in Bacteria Gene Cloning
A recombinant DNA molecule is produced. A vector must be found which in this case is a plasmid from bacteria. The plasmid must have an origin of replication, carry a gene making it resistant to a certain type of drug, and it must have one or more restriction endonuclease sites.
Step Two Gene Cloning in Bacteria
The reaction mixture for producing the recombinant DNA is introduced into the bacteria. The bacterial cells are treated with particular chemicals that make the cell membranes permeable to the DNA.
Transformation
In recombinant DNA technology, a process in which a bacterial host takes up a segment of DNA from the environment under particular experimental conditions.
Step Three of Gene Cloning in Bacteria
The bacterial cells are applied to a Petri dish containing growth media that has been supplemented with the antibiotic ampicillin and a derivative of galactose, X-gal, which causes bacterial colonies to turn blue when the bacteria is broken down by the enzyme coded for by the lacZ gene.
Step 4 of Gene Cloning in Bacteria
Bacterial colonies containing cells that have the recombinant DNA are identified using the process of elimination with markers.
Step 5 of Gene Cloning in Bacteria
Cells from the colonies that contain the recombinant DNA are selected and grown in liquid culture to produce a larger population.
Step 6 in Gene Cloning in Bacteria
The recombinant DNA molecules are isolated and purified from the bacterial cells.
Step 7 of Gene Cloning in Bacteria
A variety of analysis techniques are used to confirm that the correct recombinant DNA molecule has been made.
DNA Amplification
The process of producing large quantities of DNA from a sample.
Polymerase Chain Reaction (PCR)
An automated method for amplifying specific regions of DNA from extremely small quantities.
Step One In PCR
DNA strand to be amplified is heated to a high temperature to denature the DNA into single strands (95 degrees)
Step Two in PCR
DNA sample is cooled in the presence of two nucleotide primers; complementary to each 3’ end of the DNA fragment to be amplified (55 degrees)
Step Three in PCR
DNA sample is heated to 72 degrees-a DNA polymerase (Taq polymerase from thermophiles) begins synthesizing new DNA
Step Four of PCR
Steps 1-3 are repeated several times to make multiple copies of the DNA
Applications of PCR
- evolutionary studies
- screening for genetic defects
- crime scene DNA
Gel Electrophoresis
Tool used to separate molecules according to their mass and charge; can be used to separate fragments of DNA.
In gel electrophoresis, shorter segments of DNA move ______ than large segments which move _______.
Faster;Slower
DNA Fingerprinting
A technology used to identify individuals by analyzing the DNA sequence of certain regions of their genome.
Short Tandem Repeat (STR) Profiling
A technology used to identify individuals based on repeating short sequences of DNA in the genome hat vary in length between individuals.
DNA Sequencing
A method for determining the nucleotide sequence, base by base, of a fragment of DNA.
Dideoxy Sequencing
A method for determining the sequence of a DNA fragment using dideoxynucleotides which cause termination of DNA synthesis during the procedure.
Human Genome Project
A project that sequenced the human genome and identified all the genes within it.
Site-directed Mutagenesis
A method of specifically altering the nucleotide sequence of a region of DNA.
What is CRISPR?
- Clustered Regularly Inter-spaced Short Palindromic Repeats
- A mechanism of gene editing that can be used to alter genetic sequences
Properties of CRISPR that make it such a huge breakthrough?
- can be programmed to cut DNA at a certain sequence so possibly a sequence that contains a faulty gene
- can also insert a new DNA sequence to correct another
- relatively simple to use
Benefits of CRISPR
- can be used to possibly alter the mutations that cause genetic disorders
- can be used to alter embryos (possibly stopping a genetic disorder before the baby is born)
- fast and cheap
- can possibly target cancer cells
Ethical Issues of CRISPR
- “designer babies”
- who will have access to the technology (only people with money to pay for it?)
