Manipulating Genomes Flashcards
What is PCR used for?
Select a fragment of DNA and amplify it to produce millions of copies in a few hours
What do you need in the reaction mixture of a PCR?
DNA sample
Free nucleotides
Primers
DNA polymerase
Primer
Short piece of DNA that is complementary to the bases at the start of the fragment you want to copy
Electropheresis
Uses an electric current to separate DNA, RNA fragments, or proteins by size
How is electropheresis carried out?
- agarose gel poured into gel tray and left to solidify
- row of wells created at one end of the gel
- put gel tray into gel box and ensure wells are closest to -ve electrode
- add buffer solution to reservoirs at sides of gel box so surface of gel is covered in buffer
- add loading dye to each well to help samples sink to the bottom of wells and make them easier to see
- add diff DNA sample to each well w micropipette
- put lid on gel box and connect to power supply, causing electrical current to pass through gel
- DNA fragments are -vely charged so move through gel to +ve electrode (anode). Smaller fragments travel faster so DNA separates acc to size
- let gel run for 30 mins and turn off power. Tip off excess buffer and cover surface of gel w staining sol. Rinse w water.
How do you adapt the method of electropheresis for proteins?
Proteins can be both -vely or +vely charged, so before electropheresis they are mixed with a chemical that denatures the proteins so they all have the same charge
How to use restriction enzymes to cut out DNA fragments
- restriction enzymes recognise specific palindromic sequences (recognition sequences) and cut/digest DNA at these places
- the shape of the recognition sequence is complementary to an enzymes active site so they are specific
- you can remove DNA fragments by cutting at either end
- DNA sample is incubated w the specific restriction enzyme, which cuts the DNA out via a hydrolysis reaction
Sometimes cut leaves sticky ends
Exons
DNA that codes for protein
Introns
Large non coding sections of DNA. Spliced out of mRNA before it is translated into a polypeptide chain
Satellite DNA
Short sequences of DNA repeated many times in introns telomeres and centromeres
Minisatellites/ variable number tandem repeats (VNTRs)
Sequence of 20-50 base pairs repeated more than 50-100s of times. Occur at >1000 locations in the human genome
Microstatellite/ short tandem repeats (STRs)
Just 2-4 bases repeated 5-15 times.
Describe the steps in DNA sequencing (the capillary method
DNA sample mixed with DNA polymerase, primer, an excess of normal nucleotides, and terminator bases
- mixture placed in thermal cycler (used in PCR) that changes the temp at programmed intervals in repeated cycles.
- At 96C double strand splits to single strands.
- At 50C the primers anneal to the DNA strand
- At 60C DNA polymerase builds up new DNA strands by adding nucleotides w complementary bases to template strand
- many DNA fragments of diff lengths made
- DNA fragments separated by length using gel electrophoresis in minute capillary tubes (smallest travel fastest)
DNA sequencing
the process of determining the precise order of nucleotides within a DNA molecule
terminator bases
modified versions of the four bases (ACTG)
- no O on the OH of C-3 of deoxyribose, meaning they cannot form phosphodiester bonds with PO4 3- and form a new strand of DNA
- coloured fluorescent tags which can be detected by lasers
Describe the process of “massively parallel sequencing” or “next-generation sequencing”.
high-throughput sequencing
- instead of gel or capillaries, millions of DNA fragments attached to plastic slide called flow cell
- replicated in situ using PCR to form clusters of identical DNA fragments
- coloured terminator base still used to stop reaction so picture can be taken
Describe the reasons for developing new DNA sequencing technologies.
- quicker and easier
bioinformatics
the development of the software and computing tools needed to organise and analyse raw biological data
computational biology
using biological data to build theoretical models of biological systems, which can be used to predict outcomes
proteomics
the study and amino acid sequencing of an organism’s entire protein complement
DNA barcoding
taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to a particular species.
- section is small enough to be sequenced quickly and cheaply, yet varies enough to give clear differences btwn species
- section of mitochondrial DNA in animals
- two regions of land plant DNA in chloroplasts
Explain why new DNA sequencing methods are allowing genome-wide comparisons between individuals and between species
sequencing has become
- automated
- cheaper
- faster
computers can look at 1000s of genomes and reveal patterns in the genes we inherit and are vulnerable to
Why sequence a genome?
- species identification (DNA barcoding)
- identify the evolutionary relationships between species.
Explain how DNA barcoding allows the identification of species.
compare a section of DNA that is common to all species but varies between them, so differences in base sequence allows species to be categorised
Describe how DNA sequencing allows scientists to identify the evolutionary relationships between species.
using the basic rate of mutation and by comparing the DNA sequences of different organisms you can calculate how long ago two species evolved from a common ancestor
Explain why, in theory, knowing a DNA sequence should allow you to identify the sequence of amino acids in the protein that the DNA sequence codes for. Also, explain why, in practice, this doesn’t always provide the correct sequence of amino acids in the protein.
there are 20-25,000 protein coding genes, but a v different no. unique proteins.
some genes can code for many different proteins.
Describe 4 techniques that could be classified as “synthetic biology”. Describe the role of DNA sequencing in each technique.
> Genetic engineering
- single change in biological pathway or major genetic mod of entire organism
> Industry
- use of biosystems or parts of them in industry
eg. fixed/immobilised enzymes & drug production from microorganisms
> Synthesis of new genes to replace faulty genes
- eg. synthesise functional genes in the lab and use them to replace faulty genes in the cells of those with CF
> Synthesis of entire new organism
- eg. in 2010 creation of functioning artificial genome for a bacterium which replaced its original genome
DNA profiling
Producing an image of the patterns in the DNA of an individual. Mapping satellite regions (no. repeated DNA sections). Can help identify family relationships
exon
protein coding DNA
intron
non-protein coding DNA
locus
fixed position on a chromosome, like the position of a gene or a genetic marker
variable number tandem repeat (VNTR)/ minisatellite
within introns, telomeeres and centromeres
20-50 base pairs
repeated 50-100s of times
occur at more than 1000 locations in the human genome
short tandem repeat (STR)/ microsatellite
within introns, telomeeres and centromeres
2-4 base pairs
repeated 5-15 times
Describe how STRs vary and why they can be used to identify individuals.
appear at same positions on the chromosomes but no. repeats varies between individuals as different lengths of repeats are inherited from both parents
Name the 5 main stages in DNA profiling and describe the events in each stage
Extracting the DNA Digesting the sample Separating the DNA fragments Hybridisation Looking at evidence
MRSA
methicillin-resistant Staphylococcus aureus
Describe how DNA can be amplified using the polymerase chain reaction (PCR)
- DNA mixture heated to 95C to break the H bonds between the two strands of DNA. (dna polymerase doesn’t denature)
- mixture cooled to between 55-60C so the primers can anneal to the strands
- mixture heated to 72C so DNA polymerase can line up free nucleotides along each template strand. Complementary base pairing means complementary strands form
- two new copies of the fragment of DNA are formed and one cycle of PCR is complete
- cycle starts again and mixture is heated to 95, this time all 4 strands are used as templates
Explain the reason for the cycle of temperature changes in PCR
triggers different stages of the process
95- breaks H bonds holding strands tog
55-60 primers anneal to ends of DNA strands so repl can occur
72-75 DNA polymerase optimum temp
How many DNA molecules will be produced after 30 PCR cycles?
1 billion copies of original sample
Describe the two properties of DNA fragments that determine how far they travel in gel electrophoresis.
mass and length
Define the term “DNA probe”, explain the role of DNA probes in gel electrophoresis and describe how they can be labelled.
DNA probe is a short single-stranded section of DNA/RNA that is complementary to a known DNA sequ.
- bind to complementary strands under particular temp and pH conditions (HYBRIDISATION)
- radioactive or fluorescent
- identify microsatellite regions
- tags detected by Xray (radio) or UV (fluor) to give a pattern of bars = DNA profile
transgenic/ GMO
an organism that carries a gene from another organism
recombinant DNA
2 sources DNA; ref. sticky ends; complementary binding; H-bonds between bases; A to T and C to G; nicks in sugar-phosphate backbone sealed/AW; by ligase;
vector
means of inserting DNA form one organism into the cells of another organism
recombinant DNA
2 sources DNA; ref. sticky ends; complementary binding; H-bonds between bases; A to T and C to G; nicks in sugar-phosphate backbone sealed; by DNA ligase;
Describe the principles of genetic engineering
Isolate a gene for a desirable characteristic in one organism and place it in another organism using a suitable vector
Describe 2 ways in which a desired gene can be isolated.
> Restriction endonuclease enzymes
- cut req gene from the DNA of an organism
> Isolate mRNA for desired gene and use reverse transcriptase enzyme to produce a single strand of comp DNA (cDNA)
Explain how a plasmid can be used as a vector
- can replicate independently of chromosomal DNA
germ line cell gene therapy
inserting a healthy allele into the germ cells or a very early embryo
restriction enzyme
endonuclease; cuts DNA; with sticky or blunt ends; at, palindromic/AW/specific/4 to 6 base pair/restriction, site; from bacteria; for cutting ‘phage DNA;
