Manipulating Genomes Flashcards
Define DNA sequencing
working out the sequence of bases in a strand of DNA
Define genome
all the genetic material of an organism
Define intron
a region of non-coding DNA or RNA
Define exon
a sequence of DNA that codes for an amino acid sequence
Define DNA profiling
producing an image of the patterns in the non-coding DNA of an individual
Define polymerase chain reaction
a process by which a small sample of DNA can be amplified using specific enzymes and temperature changes
Define restriction endonucleases
enzymes that chop a strand of DNA into small pieces
Define electrophoresis
a type of chromatography that relies on the way charged particles move through a gel under the influence of an electric current and it is used to separate nucleic acid fragments or proteins
Define minisatellite
sequences of 20-25 base pairs will be repeated from 50 to several hundred times (variable number of Tandem repeats VNTR’s)
Define microsatellite
a region of 2-4 bases repeated 5-15 times (short Tandem repeats STR’s)
Define telomere
the distinctive cap-like structure present at the end of each strand of DNA and provide protection against DNA damage
Define centromere
the region at which 2 chromatids are held together
What is the purpose of PCR?
it can take a fragment of DNA and produce millions of copies in a short period of time - it is a method of amplifying DNA
What is in a PCR reaction mixture?
DNA, free nucleotides, primers and Taq polymerase
What is the role of DNA primers in PCR?
They are artificial complimentary sequences of DNA that latch onto the desired sequences so that DNA polymerase can attach to the strand and begin synthesising a complimentary strand.
What is the role of Taq DNA polymerase in PCR?
It is a temperature resistant enzyme responsible for DNA replication by assembling nucleotides along a complimentary strand.
What is the role of fluorescent markers in PCR?
Allows you to see how much DNA has been amplified
What are primers complimentary to in PCR?
primers are complimentary to the sequence at the beginning of the piece of DNA you want to look at
What temperature does the denaturing stage in PCR take place at?
95 degrees C
What temperature does the annealing stage of PCR take place at?
55 degrees C
What temperature does the extension stage of PCR take place at?
72 degrees C
What happens in the denaturing stage of PCR?
this stage breaks the hydrogen bonds allowing the DNA strands to separate
What happens during the annealing stage of PCR?
the solution is cooled enough to allow the DNA primers to attach at the start of the STR’s
What happens during the extension stage of PCR?
Taq polymerase attaches the nucleotides to synthesise a complimentary strand
What are the ingredients in DNA sequencing capillary (chain termination) method?
- DNA strand/fragment to sequence
- terminator bases (A T C G)
- primer
- DNA polymerase
- free nucleotides (A T C G)
What are terminator bases also known as?
dioxynucleotides
What are the steps in DNA sequencing capillary (chain termination) method?
1, starting components mixed together
2, mixture heated to 95 degrees C to separate strands
3, cooled to 50 degrees C to allow the primers to anneal to the DNA
4, heated to 60 degrees C - DNA polymerase synthesises complimentary strands of DNA. If a terminator base is added then the DNA polymerase stops. This will result in an incomplete DNA strand
5, DNA is read in size order with the terminator nucleotide having a fluorescent tag in a base specific colour
When did the human genome project start?
1990
When was the first draft of the human genome project completed?
2000
When was the human genome project published?
2003
Why are sequenced genes and genomes useful?
- predicting polypeptide sequences
- allows artificially made proteins to be produced (synthetic biology)
- designing biological systems and molecules that don’t exist in humans but may be beneficial
- DNA can be created from scratch
- synthesis of new genes to replace faulty ones
Explain why a temperature of 72-75 degrees C is used for synthesising a new strand (in PCR)
This is the optimum temperature for Taq polymerase to work at.
Taq polymerase allows higher rate of DNA replication.
Taq polymerase is able to withstand higher temperatures than normal DNA polymerase.
On a DNA profile suggest why some loci have two peaks but some have only one.
- double peaks are heterozygous/have different alleles
- single peaks are homozygous/have the same allele
What is a restriction endonuclease?
an enzyme that cuts DNA at specific sequences, leaving sticky ends
What is ligase?
an enzyme that glues sticky ends together by forming phosphodiester bonds
Why is electroporation used in genetic engineering of bacteria?
to increase the permeability of the bacterial cell membrane to increase the chance of plasmid uptake
List the three stages in PCR and the temperatures involved.
1, strand separation (denaturation) : 95 degrees C
2, addition of primers and binding (annealing) : 55 degrees C
3, DNA strand synthesis (extension) : 72 degrees C
What are VNTRs?
variable number tandem repeats
How do VNTRs enable DNA profiling to identify individuals?
the probability of two people having the same VNTRs is very low
During DNA sequencing, why is the mixture heated to 95 degrees C at the beginning of a cycle?
to separate the DNA double strands
Why are terminator nucleotides used in DNA sequencing?
so the DNA strands are truncated, to make the DNA fragments different lengths
How are the DNA strands produced through sequencing separated?
by gel electrophoresis
Does electrophoresis separate DNA fragments by size or charge?
by size
How are the fragments separated by gel electrophoresis?
by an electric current pulling the charged fragments through the gel
What charge do DNA fragments carry and to which terminal of the gel are they pulled?
negatively charged, pulled towards the positive terminal of the gel
