Recombinant DNA technology Flashcards
How can plasmids be genetically modified and inserted into bacteria
- isolate the wanted gene from the DNA
- using restriction endonuclease to leave complementary sticky ends on the selected gene and the plasmid
- insert the gene into plasmid using ligase enzyme with the addition of a marker e.g. antibiotic resistance
- put the plasmid containing the recombinant DNA back into the bacteria by heating then cooling then heating
- grow on a medium where marker would be expressed e.g. containing antibiotics
- bacteria that grows will have be antibiotic resistant and contain the desired gene
Name the type of enzyme used to cut open the plasmid
restriction endonuclease
Name the type of enzyme used to insert the gene onto the plasmid
ligase
Describe the process of genetic fingerprinting
- DNA is cut into fragments using a restriction enzyme
- DNA fragments are separated according to size by gel electrophoresis under electrical influence
- gel is then immersed into alkali to separate the double strands into single strands (southern blotting) and then transferred onto a nylon membrane
- radioactive/fluorescent DNA probs are used to bind to the complementary base sequence of the VNTRs under specific temperature and pH conditions
- nylon film can be x-rayed and the probs will be exposed, fluorescent probe can be located visually
What is the process of PCR (Polymerase Chain Reaction)
increases the amount of DMA in a sample by:
- DNA strand is separated and the DNA fragments, primers and DNA polymerase are placed in a vessel, and heated to 95°C
- the mixture is cooled to 55°C, causing the primers to anneal (join)to their complementary bases at the ends of the DNA fragment
- the synthesis of DNA happens when the temperature is increased to 72°C which is the optimal temperature for the DNA polymerase to add complementary nucleotides to the DNA strands
What is a primer of the PCR
- short sequences of nucleotides that have a set of bases complementary to those at one end of the two DNA fragments
- provide a starting point for the DNA polymerase to begin copying the DNA
- prevent two separate DNA strands from rejoining
What is in vitro cloning
using method of PCR that is rapid and does not require living cells
What is in vivo cloning
isolating the gene of interest and use of gene machine
What is meant by the term Variable Number Tandem Repeat (VNTR)
highly repetitive sequences of DNA bases
How does the structure of a genome allow it to be used for genetic fingerprinting
- VNTRs differ in size/length between different individuals
- VNTRs occur in lots of different places in the genome between different individuals
- the difference in size and location of VNTRs creates a genetic fingerprint unique to the individual which can be used to identify them
What is southern blotting used for
to separate double-stranded DNA, to make it single-stranded for the DNA probe to bind
How is a gene made using a gene machine
- the amino acid sequence os determined
- the nucleotide triplets are worked out and the base sequence is fed into a computer
- the computer designs gly
Describe the following stem cells: Totipotent Pluripotent Multipotent Unipotent
totipotent - can differentiate into any type of cell, found in early embryo
pluripotent - can differentiate into almost any type of cell, found in embryonic and fetal stem cells
multipotent - can differentiate into limited number of specialised cells, found in adult and umbilical cord blood stem cells
unipotent - can differentiate into a single type of cell, found in adult tissue
What are induced pluripotent stem cells (iPS cells)
- they are very similar to embryonic stem cells (pluripotent cells) however they can potentially divide indefinitely
- can overcome ethical issues surrounding use of embryonic stem cells
- can be used to regrow tissues
What is decreased acetylation
- acetyl group is removed from molecule
- increases positive charges on histones and therefore increases attraction to the phosphate groups of DNA
- DNA becomes less accessible to transcription factors to initiate mRNA productions