Flashcards in 6.3 - Manipulating Genomes Deck (55)
What do restriction enzymes do?
Restriction enzymes are used to cut out DNA fragments. They cut DNA at specific palindromic base sequences (by hydrolysis). Leave sticky or blunt ends.
What is a palindromic sequence?
The sequences consist of short antiparallel base pairs (about 4-6 base pairs). The base pairs read the same in opposite directions.
What is a sticky end and why are they useful in genetic engineering?
When restriction enzymes cut in a staggered way leaving small tails of unpaired bases at each end of the fragment. These unpaired bases can be used to bind (anneal) to another DNA fragment that has sticky ends with complementary sequences.
In simple terms how is a transgenic organism produced?
A gene(s) from one organism is isolated and placed into another organism using a suitable vector.
What is a vector (GM)?
A carrier – transfers DNA into a cell.
They can be a plasmid (small, circular molecules of bacterial DNA) or bacteriophages (viruses that infect bacteria).
Describe the process of genetic engineering.
The DNA fragment containing the desired gene is isolated using restriction enzymes.
The vector (usually a plasmid) is cut open using the same restriction enzyme.
In this way the sticky ends of the DNA fragment & the vector are complementary.
DNA ligase anneals the vector and DNA fragment (ligation) – forming recombinant DNA.
A plasmid vector then transfers the gene into the bacteria using electroporation.
(a bacteriophage vector will infect the bacterium by injecting its DNA into it & integrate the phage DNA into the bacterial DNA)
What is electroporation?
A high voltage pulse of electricity is applied to the host (bacterial) cell to disrupt the membrane – making it more porous to encourage uptake of plasmid vector.
What is the new combination of DNA called when you have vector DNA + DNA fragment?
What are genetically engineered cells called that take up vectors containing the desired gene?
A transformed cell/organism.
What does PCR stand for?
Polymerase chain reaction.
What is PCR used for?
A DNA fragment (often containing the desired gene) can be amplified to produce millions of copies in just a few hours.
Describe the process of PCR.
a) A reaction mixture is set up containing the DNA sample, free DNA nucleotides, primers and DNA polymerase.
b) Mixture heated to 95°C – to separate the DNA strands by breaking the hydrogen bonds, resulting in two template strands.
c) Mixture cooled to 55°C – primers to anneal (join) to each template strand.
d) Mixture heated to 72°C – DNA polymerase joins the lined up complementary nucleotides alongside each template strand to form two new complementary DNA
e) Cycle starts again, this time all four strands (two original & two new) are used as templates.
f) The cycle is repeated many times, each cycle doubles the amount of DNA.
What is unusual about the DNA (Taq) polymerase used in PCR?
It does not denature at the high temperatures used in PCR.
Why is PCR useful and what are some applications of PCR?
PCR amplifies small samples of DNA and so is useful to increase the amount of DNA available for analysis.
What is electrophoresis?
An electrical current is used to separate out DNA fragments, RNA fragments or proteins depending on their size.
Describe the process of electrophoresis of DNA.
Cover the agarose gel with a buffer solution.
Fluorescently labelled DNA samples have loading dye added to help them sink to the bottom of the wells in the gel.
Add DNA samples to wells at the negative electrode end.
Pass an electrical current through the gel for about 30 minutes.
The smaller the fragment the faster it travels through the gel.
Why is a buffer used in electrophoresis?
To provide ions that carry a current and to maintain the pH at a relatively constant value.
Why are the DNA samples placed at the negative electrode end?
DNA is negatively charged and so moves toward the positive electrode (anode) at the other end of the gel.
Why is a fluorescent tag added to DNA samples prior to loading on the gel?
For visualisation of DNA samples under UV light after electrophoresis to ascertain how far the bands of DNA have travelled.
What is a DNA ladder used for?
To compare a set of known banding patterns to identify the length of sample DNA fragments.
Why can this basic method (fluorescence) be used for electrophoresis of RNA samples, but not protein samples?
RNA is negatively charged and so treated the same as DNA.
Proteins can be positive or negatively charged. They must be denatured before electrophoresis so they are all the same charge.
What fact is DNA profiling based on?
The fact that the DNA of every individual, except identical twins, is unique.
Within the introns are repetitive, non-coding base sequences called short tandem repeats (STRs).
The number of times these sequences are repeated differs from person to person, so the length of these sequences differs from person to person.
Describe DNA profiling.
DNA profiling compares the number of times a STR sequence is repeated AND the different places it is repeated in an individual’s genome.
The probability of two individuals having the same length of repetitive sequence is very low.
In modern DNA profiling, 13 STRs are analysed simultaneously.
How is a DNA profile made?
Sample of DNA obtained (saliva/blood), cut using restriction enzymes (to cut out the exons) and amplified by PCR.
Fluorescent tag added to DNA fragments.
Fragments undergo electrophoresis. Viewed under UV light.
Compare to known banding patterns to identify the length (DNA ladder).
How can DNA profiling be used?
Preimplantation genetic haplotyping (PGH) – screen embryos for cystic fibrosis & Huntingtons disease before implantation in the uterus.
Why can red blood cells not be used for DNA profiling?
They do not contain a nucleus and hence have no DNA to profile.
What is gene sequencing & what is the traditional method called?
Used to determine the order of bases in a section of DNA (gene) (up to 750 base pairs long). Carried out by chain termination method (Sanger method).
Describe the Sanger method for gene sequencing.
The following mixture is added to 4 separate tubes:
A single-stranded DNA template (the DNA to be sequenced).
DNA polymerase (enzyme that joins DNA together).
Lots of DNA primer (short pieces of DNA) – to start process of DNA synthesis. Also carry a fluorescent labelled tag for identification of DNA fragment produced.
Fluorescently-labelled modified nucleotide –they cannot join to normal nucleotides so acts as chain terminators, ending DNA synthesis.
Free DNA nucleotides + one of the 4 modified nucleotides.
The binding of the (modified) terminator nucleotides (dideoxy nucleotides) is random resulting in strands of varying in lengths.
These strands can be sorted by size using gel electrophoresis.
Which way do you read the gel to sequence the new strand?
From the smallest fragment (bottom or positive electrode) to the largest fragment.