module 14-15 Flashcards
what is gel electrophoresis??
how does it work?
-a technique used to separate fragments of DNA based on size and charge.
-each sample (mixture of DNA fragments) are placed separately in wells on the negatively charged end.
-these wells are near one end of a thin slab of agarose gel. the gel is set into a small plastic support and immersed in a buffered solution with electrodes at each end.
-the electric current is turned on and negatively charged fragments move toward the positively charged anode. smaller and more negative fragments move through the gel quicker.
-after the current is turned off, DNA-binding dye is added which becomes pink in UV light. each pink band corresponds to many DNA molecules of the same length.
-at the bottom of the gel is a set of restriction fragments of known size. they are used for comparison with samples of unknown length.
in gel electrophoresis, what fragments move through the gel faster and why?
-fragments that are more negatively charged are more attracted to the positively charged end and move through the agarose gel quicker.
-fragments that are smaller pass through the matrix of tunnels created by the agarose gel faster.
what are the steps involved in Sanger’s (1977) chain termination method?
- mix together single strand DNA, primer, excess of dNTPs, and a small amount of ddNTPs, and DNA polymerase.
- terminates strand whenever it is added, each time they are fragmented, termination occurs.
3.seperate type for each nucleotide. each nucleotide is radioactively labelled. - read off sequence of bases starting from bottom of gel
- each vertical column corresponds to one of the four bases.
what are the steps involved in modern-day chain termination?
1.a fragment of DNA is denatured into single strands then incubated in test tube containing necessary components for DNA synthesis (primer designed to join to known 3’ end of template strand, DNA polymerase, four dNTPs and four ddNTPs (each flagged with specific fluorescent molecule))
2.synthesis of new strand starts at 3’ end of primer, continues until ddNTP is inserted instead of the equivalent dNTP. incorporated ddNTP prevents further elongation.
- eventually a set of labelled strands of each possible length are generated. with the colour of the tag representing the last nucleotide in the sequence.
- the labelled strands go through the process of gel electrophoresis (capillary tube is used in place of gel).
5.the capillary tube’s small diameter allows a fluorescent detector to sense the colour of each tag as the strand comes through.
what are the steps involved in next generation sequencing?
1.genomic DNA is fragmented, fragments of 400-1,000 bp selected.
2.each fragment is isolated with a bead in an aqueous solution.
3.fragments are copied by PCR. all 5’ ends of strands are ‘captured’ by bead. eventually 10^6 identical copies of the template strand are attached to the bead
4.the bead is placed in a small well along with DNA polymerase, and primers that can hybridise to the 3’ end of the template strand.
5. in each well, if the next base on the template strand is complementary to the added nucleotide, the nucleotide is added to the growing strand. this releases PPi which causes a flash of light that is recorded.
6.the nucleotide is then washed off, the a different nucleotide is added. if the nucleotide isn’t complementary to the template base, joining doesn’t occur and no flash is seen.
7. this process is repeated until every fragment has a complete complementary strand. the pattern of flashes reveals the sequence of the original fragment in each well.
Describe the denaturing phase of polymerase chain reaction
Denaturing - Heat DNA to 90oC to separate the DNA into single strands by breaking hydrogen bonds between complementary bases.
Describe the primer annealing phase of the polymerase chain reaction
DNA Primer Annealing - Cool to approximately 50oC to attach/anneal DNA primers to the single-stranded DNA.
Describe the elongation phase of the polymerase chain reaction
Elongation - Heated to approximately 72oC - DNA Taq polymerase copies the single DNA strands in a 3’ to 5’ direction using complementary base pairing.
State the purpose of the polymerase chain reaction
To amplify the DNA so that there is enough to analyse
Name the fours stages of PCR
Denature/Dehydridise
Primers Anneal
Extension/Elongation
Repeat 35+
Outline the four stages of PCR
Denaturing - Heat DNA to approximately 90C to separate the DNA strands.
Primer annealing - Cool to attach approximately 50C to attach primers at the 3’ end of each DNA strand.
Extension (elongation) - Heat to 70C so Taq polymerase can copy strands in a 3’to 5’ direction.
Repeats - Process is repeated 35 times.
DNA primers are
Short single-stranded DNA fragments that attach to DNA to allow the binding of DNA polymerase
Reverse transcriptase is a
enzyme that copies mRNA into copy DNA
State the function of an restriction endonuclease/restriction enzyme
Bacterial enzyme that cuts DNA at a specific recognition sequence/site
A plasmid is a
Small ring of bacterial DNA can be used as a vector for DNA recombination and insertion
why are STRs useful in DNA profiling?
PCR with flanking primers can detect repeat length variation and allow sensitive and rapid production of DNA fingerprints.
STRs can be identified through this process and as STRs are hypervariable, DNA fingerprints are formed.
The bands containing STRs ran through PCR can be compared with other Individuals (useful with paternity test, genetic profiling and forensics).
what is a ddNTP?
dideoxyribonucleoside triphosphate.
a type of nucleoside triphosphate used in DNA sequencing methods used to terminate strands.
does not have a hydroxyl group at its 3’ end unlike
deoxyribonucleoside triphosphate.
what is a dNTP?
deoxyribonucleoside triphosphate.
a type of nucleoside triphosphate used in DNA sequencing methods used to terminate strands.
