Flashcards in Gene Technology Deck (54)
What is a Palindromic site?
its read the same forwards and backwards
(eg) CTTAAG to GAATTC)
What is the role of restriction enzymes?
They cut DNA at specific Palindromic sites called restriction sites
What does using a restriction enzyme leave DNA with?
sticky ends (unpaired bases)
What does reverse transcriptase do?
mRNA -------> cDNA
What does cDNA not have?
introns (made from mRNA post splicing)
What enzyme does DNA ------> mRNA?
Describe Artificial Synthesis of a gene.
use a gene machine to make DNA from scratch, making oligonucleotides, join theses together to make a synthetic gene.
What is an oligonucleotide?
a sequence of around 25 nucleotides
What are the three ways we can isolate target genes?
gene machine, reverse transcriptase or restriction enzymes
What is PCR used for?
to amplify a gene
What are the 4 components of PCR?
DNA sample, free nucleotides, primers, DNA polymerase
describe the 3 steps of PCR
heat up to 95 degrees
cool to 50 degrees
heat to 70 degrees
Why do we heat the sample to 95 degrees
break the H+ bonds between so they are single stranded
Why do we cool the sample to 50 degrees
allows the primer to attach by complementary base pairing to the exposed strand - making a double strand - so DNA polymerase can bind to the double stranded section
Why do we heat the sample back up to 70 degrees
there is a higher kinetic energy increasing enzyme action
why can the DNA polymerase work at high temperatures?
extracted from thermophillic bacteria
What is a primer?
Short sequence of DNA which is complementary to the target DNA
Name three ways we can isolate a target gene
restriction enzymes, Gene machine, reverse transcriptase
What charge is DNA?
Describe the process of electrophoresis
attach a fluorescent label to DNA
put labelled DNA fragments into the well at the negative end
turn on current
DNA is attracted to the positive electrode
How can we distinguish the length of DNA from electrophoresis?
smaller fragments move faster (therefore further over any given time)
How can you calibrate the scale in electrophoresis?
use pieces of DNA of a known length
How can we insert the isolated target gene into a vector?
use the same restriction enzyme so that the sticky ends are complementary
DNA ligase makes recombinant DNA
How can the vector be inserted into the plasmid?
ice cold calcium chloride and a heat shock to make a transgenic/ transformed organism
How can scientists identify transformed/transgenic organisms?
marker genes, fluorescence, antibiotic resistance
Why do we then culture the transformed/ transgenic organism (bacteria)
the organism transforms/ translates the recombinant DNA
to make desired protein eg) insulin
What is gene therapy?
altering faulty alleles that cause genetic disease
How can we silence a genetic disorder caused by a dominant allele? (eg) Huntingtons)
sufferer is heterozygous
use a vector to add a DNA fragment into dominant allele
this prevents transcription
recessive allele expressed
How can we silence a recessive allele disease (eg) cystic fibrosis)
sufferer is homozygous
use a vector to add the functional allele to DNA
dominant allele will be expressed