Module 6 Section 4: Manipulating Genomes Flashcards
What are the different techniques needed to study genes
Cutting out DNA fragments using restriction enzymes
Polymerase chain reaction (PCR)
DNA sequencing
Gel electrophoresis
How is PCR used
Can be used to select a fragment of DNA (containing the gene or area of DNA you’re interested in) and amplify it to produce millions of copies
Occurs in a few hours
Full process of PCR
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA.
DNA polymerase doesn’t denature even at this high temperature
The mixture is then cooled to between
50 and 65 °C so that the primers can bind (anneal) to the strands.
The reaction mixture is heated to 72 °C so DNA polymerase can work.
The DNA polymerase lines up free DNA nucleotides alongside each template strand.
Complementary base pairing means new complementary strands are formed.
Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
The cycle starts again, with the mixture being heated to 95 °C and this time all four strands (two original and two new) are used as templates
How are primers and DNA polymerase used in PCR
Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
DNA polymerase is an enzyme that creates new DNA strands.
Why is it important that DNA polymerase doesn’t denature at the temperatures PCR uses
It means many cycles of PCR can be carried out without having to use new enzymes each time
How much DNA can a PCR cycle produce
Each PCR cycle doubles the amount of DNA,
e.g. 1st cycle = 2 × 2 = 4 DNA fragments, 2nd cycle = 4 × 2 = 8 DNA fragments, 3rd cycle = 8 × 2 = 16 DNA fragments
What is electrophoresis used for
Uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size
How to set up apparatus for electrophoresis
Pour agarose gel into a gel tray and leave to solidify.
A row of wells is created at one end of the gel.
Put the gel tray into a gel box (or tank).
Make sure end of the gel tray with the wells is closest to the negative electrode on the gel box.
Add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution.
How to load DNA samples into wells
Use micropipette to add the same volume of loading dye to DNA fragments (from PCR or restriction enzymes)
Loading dye helps samples sink to the bottom of the wells to make them easier to see
Add 10μl of DNA sample to first well
Make sure tip of micropipette is in buffer solution and just above opening of the well
Don’t stick tip too far into well as it may pierce bottom
Repeat process and add same volume of the other DNA samples to other wells in the gel
Clean tip of micropipette each time
Record the DNA added to each well
How to carry out electrophoresis
Put lid on box and connect leads from gel box to power supply
Pass 100V through the gel
Run gel for 30mins or until dye is 2cm from end of the gel
Remove gel tray from the gel box and tip off excess buffer solution
Stain DNA fragments by covering surface of the gel with staining solution then rinsing gel with water
Bands of the different DNA fragments will now be visible
How to measure the size of the DNA fragments
Size of DNA fragment is measured in bases
E.g. ATCC = 4 bases or base pairs
1000 bases is one kilobase (1 kb)
How do DNA fragments separate in electrophoresis
DNA fragments are negatively charged so they’ll move through the gel towards positive electrode at far end of the gel (anode)
Small DNA fragments move faster and travel further through gel
Means DNA fragments separate according to size
How are RNA fragments separated using electrophoresis
This follows the same basic method as for DNA fragments
How are proteins separated using electrophoresis
Proteins can be positively charged or negatively charged
Before they undergo electrophoresis, they’re mixed with a chemical that denatures the proteins so they all have the same charge.
Uses of electrophoresis of proteins
e.g. to identify the proteins present in urine or blood samples, which may help to diagnose disease.
What are palindromic sequences of nucleotides
These sequences consist of the same order of bases when read backwards on the opposite strand
What are restriction enzymes
Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
They cut the DNA via hydrolysis reactions
Also called restriction endonucleases
How can you choose what type of DNA fragment are formed
Can choose between different restriction enzymes
They can cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site.
Results in different fragments forming
E.g. the restriction enzyme EcoRI cuts at GAATTC, but HindIll cuts at AAGCTT.
How to use a restriction enzyme in a lab
The DNA sample is incubated with the specific restriction enzyme
What do you need in order to completely separate a fragment from the whole chain
If recognition sequences are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA.
How are restriction enzymes used to bind different fragments together
Sometimes the cut leaves sticky ends
These are small tails of unpaired bases at each end of the fragment.
Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
What is a DNA profile
Some of an organism’s genome consists of repetitive, non-coding base sequences which are called short tandem repeats and are within satellite DNA
These are sequences that don’t code for proteins and repeat over and over
E.g. introns, telomeres (caps and tails), centromeres
The number of repetitions of non-coding sequences differs from person to person, so the length of these sequences in nucleotides differs too.
This is their DNA profile
PCR is used to increase DNA sample size
The number of times a short tandem repeat is repeated at different, specific places (loci) in a person’s genome (and so the number of nucleotides there) can be analysed using electrophoresis.
This creates a DNA profile.
Where is DNA profiling useful
PCR and DNA profiling is performed on traces of DNA left at the crime scene
eg. blood, semen, skin cells, saliva or hair
DNA profile is compared to a sample taken from a suspect or identified using criminal data base
Can be used to prove paternity of a child
Can be used in immigration cases to prove or disprove family relationships
DNA profiling is used to identify which species an organism belongs to
Used to demonstrate evolutionary relationships between different species
Can be used to identify individuals who are at risk of developing particular diseases
Certain non-coding microsatellites, or the repeating patterns they make, have been found to be associated with an increased risk or particular disease E.g. Huntington’s disease, sickle cell anaemia
Process of DNA sequencing
DNA for sequencing is mixed with a primer, DNA polymerase, a excess of normal nucleotides (containing bases A, T, C, G) and terminator bases
Mixture undergoes PCR in thermal cycler
At 72°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template
Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated since no more bases can be added
As the chain-terminating bases are present in lower amount and are added at random, this results in many DNA fragments of different lengths depending on where the terminating bases have been added
All possible DNA chains will be produced after many cycles as the reaction is stopped at every base
Fragments separated according to their length by capillary sequencing which is like gel electrophoresis in small capillary tubes
Final base is identified on each strand by fluorescent markers
Lasers detect different colours which shows the order of the sequence
The order of bases in capillary tubes shows the sequence of the complementary strand of DNA which is used to determine the sequence of the original DNA strand
How whole genomes be studied quickly
Chain termination technique still used but the tube contains all the modified nucleotides each with a fluorescent label
The machine reads the sequence out
High throughput sequencing can read 1000 times more bases in a given time
How does high throughput pyrosequencing work
Section of DNA cut into fragments, split into single strands and a strand from each fragment is attached to a small bead
PCR used to amplify fragments on each bead
Each bead put into separate well
Free nucleotides added to the wells attach to the DNA strands via complementary base pairing
Wells also contain specific enzymes which emit light when bases are added to DNA strand
Computers analyse occurrence and intensities of the light emitted in the different wells and process the information to interpret the DNA sequence
Technique can sequence about 400 million bases in a 10 hour period
Overview of process of how chain termination method is used to study whole genomes
Genome fragmented
Fragments inserted into BACs
DNA extracted from colony
DNA sequenced using chain termination method
Fragments put back in order
First step of preparing genome to be sequenced: BACs
Chain termination technique can only sequence fragments up to 750bp long
Genome is cut into smaller fragments (about 100 000 bp) using restriction enzymes.
Fragments are inserted into bacterial artificial chromosomes (BACS)
The BACs are then inserted into bacteria
Each bacterium contains a BAC with a different DNA fragment
Second step of preparing genome to be sequenced: DNA extracted from bacteria
The bacteria divide to create colonies of cloned (identical) cells that all contain a specific DNA fragment.
Together the different colonies make a complete genomic DNA library.
DNA is extracted from each colony and cut up using restriction enzymes (to make fragments small enough to sequence)
Produces overlapping pieces of DNA.
Third step of sequencing whole genomes: fragments sequenced
Each piece of DNA is extracted from BAC and sequenced using the chain-termination method (including terminator base types of all nucleotides)
Fragments separated out according to size with electrophoresis, fragments differ by 1 base each
Electrophoresis sheet then put through computer where laser reads the bases (by radioactive tag of fluorescent marker) and creates base sequence
Finally the DNA fragments from all the BACs are read out by computers, to complete the entire genome.
What are bacterial artificial chromosomes
These are man-made plasmids
Fragments are inserted into these BACs which are inserted into bacteria to create colonies of different fragments
Not needed specifically to sequence genome
Just there to provide genomic library so the fragments are to hand if needed
What can you deduce from sequencing a gene
The sequence of amino acids the gene codes for
Used to find primary structure of polypeptide
What is synthetic biology
This is how biological molecules are made through predicting the structure of a protein derived from a gene that has been sequenced
4 steps or genetic engineering
- Isolate the gene
- Package the gene (in a vector)
- Transfer the vector to the recipient cell
- Recipient cell expresses the gene
Processes of isolating the gene in genetic engineering
Use DNA probe to identify the gene then use restriction enzymes to cut the gene out
The restriction enzymes used are specific to DNA strands which have a certain base sequence
DNA fragments are left with sticky ends
Harvest mRNA from cells where it is being expressed
Use reverse transcriptase to convert mRNA to cDNA (complementary DNA)
Add primers and polymerase to build double stranded DNA
How do scientists identify if transformation has taken place in genetic engineering
Plasmids that are used as vectors usually have a marker gene
May have a gene for antibiotic resistance which enables scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in a media containing antibiotic
After recombinant DNA is formed the vector plasmids are given second marker gene to show plasmid contains recombinant gene
If DNA fragment is inserted successfully the marker gene will not function indicating process has worked
Marker genes can be for antibiotic resistant, fluorescence or enzyme which causes colour change
