chapter 19 : gene tech pt1 (till Microarray) Flashcards
define recombinant DNA :
recombinant DNA rDNA is DNA thats altered by genetic engineering and which now contains lengths of nucleotides from two diff organisms
what is genetic engineering?
genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism and that thsi may involve transferring a gene into an organism so that the gene is expressed
how is the gene that is to be transferred obtained ?
- extracted from the DAN of a donor host
- Synthesized from mRNA of a donor host
- Synthesized chemically from nucleotides
what are the tools needed for a gene technologist ?
- enzymes : such as restriction endonuclease, DNA ligase, reverse
- transcriptase and DNA polymerase.
- vectors : plasmids and viruses
- genes coding for easily identifiable substances that go and be used as markers
what are restriction enzymes ?
- enzyme from bacteria : which recognizes and breakdown the DNA of invading viruses known as bacteriophages (phage)
- these enzymes cut phage into smaller pieces at the sugar phosphate backbone of DNA at specific places within molecule
- known as restriction box restricts viral infection, ‘endo’ bcoz cuts within
what is the role of restriction endonucleas?
- role of restriction endonuclease is to restrict a viral infection
- each restriction enzyme bunds at target cell
- cuts at that site
how is bacterial DNA protected /arent affected by restriction enzymes ?
by chemical markers or by not having target sites
what are target sites ?
they are specific sequences of bases
what are examples of such target sites ?
EcoRI …. 5’-G / AATTC-3’ …. sticky ends
——–
3’-CTTAA / G-5’
what are sticky ends ?
- sticky ends are short unpaired bases
- can easily firm hydrogen bonds with complementary sequences
how is gene synthesized chemically from nucleotide
S
- by choosing codons for the amino acid sequence that they need.
- The sequence of nucleotides is held in a computer that directs the synthesis of short fragments of DNA.
- These fragments are then joined together to make a longer sequence of nucleotides that can be inserted into plasmids for use in genetic engineering.
what is PCR (polymerase chain reaction)?
- it is an automated method of making multiple identical copies (cloning) of a tiny sample of DNA
- PCR involves exposure of DNA to repeating sequence of different temperature s , allowing enzymes to work.
describe an overview of gene transfer
*1 The gene that is required is identified. It may be cut from a chromosome, made from mRNA by reverse transcription or synthesized from nucleotides.
*2 Multiple copies of the gene are made using the technique known as the polymerase chain reaction (PCR).
*3 The gene is inserted into a vector which delivers the gene to the cells of the organism. Examples of vectors are plasmids, viruses and liposomes.
*4. The vector takes the gene into the cells.
*5. The cells that have the new gene are identified and cloned.
what is a primer ?
This is a short length of DNA, often about 20 base pairs long, that has a base sequence complementary to the start of the part of the DNA strand that is to be copied.
describe PCR process
1- the DNA is denatured usualy by heating it at (95’) which separates the DNA molecule into two strands leaving bases exposed
2- DNA polymerase then used to build new strands of DNA against exposed ones. PRIMER STARTS the process (20 base pair length of dna complementary to the start of the dna strand to be copied.)
3- PRIMER attaches at start of DNA strand, (annealing a 65’) DNA POLYMERASE nucleotides along the rest of DNA strand.
4- DNA is copied, then mixture is heated again (72’) to separate strands into 2, building up complete new DNA strands using DNA POLYMERASE
