Chapter 21 - Manipulating Genomes Flashcards
Which end does DNA grow from?
only grows from 3’ end
strands
➜ Lagging strands:
◦ AKA Okazaki fragments (short sections of DNA) - allows DNA poly to synthesis lagging strand
◦ separated into chunks
➜ Leaving strand:
◦ completely replicated as one strand
Producing a DNA profile
- Extract the DNA
- PCR to amplify
- Digest sample via restriction endonucleases
- Separate DNA fragments - electrophoresis
- Hybridisation
- DNA sequencing
- DNA profiling!!!!
PCR
Polymerase chain reaction
➜ amplification of DNA outside of the body
➜ AKA in vitro method of DNA amplification
➜ produces large quantities from small sample (why? - crime scene, single drop of blood and imagine they fuck up the dna testing so they amplify DNA so theres lots of dna to mess up)
requirements of PCR
➜ Target DNA/RNA
➜ teeny weeny test tube known as a vial
➜ DNA polymerase - from a bacteria that is found in hot springs so that it won’t denature at high temp (Taq polymerase) as first stage of PCR is at high temp
➜ Forward and reverse primers - short sequences of single stranded DNA (ssDNA) that have base sequences complementary to the 3’ end of DNA/RNA being copied
➜ Free nucleotides
➜ Original DNA strand
➜ buffer solution - to provide optimum pH for reaction to occur in
Process of PCR
➜ uses a thermal cycler where the DNA sample, free nucleotides, primers and DNA polymerase is added into a vial
➜ It is then heated at around 95℃ to break the H bonds which forms 2 strands
◦ remember DNA poly doesn’t denature due to it being from hot springs!
◦ therefore this DNA poly can be reused
➜ Mixture is then cooled to around 50 to 60℃ so that primers can anneal (bind) to each 3’ end of single strands of DNA
➜ The mixture is then heated again to 72℃ as this is optimum temp for Taq poly (DNA poly from hot springs)
➜ The Taq line up free DNA nucleotides along the template strand and allow for complementary base pairing (start at end with primer and proceed in 5’ to 3’ direction)
➜ 2 new copies of fragment DNA formed and this is one cycle of PCR
➜ Process is repeated though heating but all 4 strands used this time (2 og and 2 new) as templates
➜ each PCR doubles it so it goes from 2 becoming 4 to 8 to 16 etc
➜ DNA ligase catalyses the formation of phosphodiester bonds (only lagging strand) in the DNA backbone
Applications of PCR
Tissue typing
➜ donor and recipient tissue can be types prior to transpantation to reduce risk of rejection
Applications of PCR
Detection of oncogenes
➜ if the type of mutation involved in a specific patients cancer is found then meds can be tailored to that patient
Applications of PCR
Detecting mutations
➜ sample of DNA is analysed for the allele with the disease
➜ parents can be tested to see if they carry a recessive allele that can lead to a genetic disease
Applications of PCR
Identifying viral infections
➜ sensitive PCR tests can detect small quantities of viral genome amongst host cells DNA
Applications of PCR
Forensics
➜ small quantities of DNA can be aplified for DNA profiling to identify criminals
➜ again the idea that most crime scenes dont have half a gallon of blood there to test loads !!
Applications of PCR
Research
➜ amplifying DNA from extinct ancient sources for analysis and sequences
Applications of PCR
Spread of infectious disease
➜ the spread of pathogens in a population can be monitored
restriction endonucleases
restriction enzyme
➜ used to cut the DNA into fragments
➜ diff restriction enzymes cut DNA at diff base sequences so scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions (regions found in the non-coding part of DNA)
Electrophoresis
➜ uses an electrical current to separate out DNA fragments depending on size
➜ occurs as DNA is negatively charged due to phosphate group and so DNA frags will move through pores of gel towards positive end (anode) where electrical current is applied
➜ different size molecules will move through agarose at diff rates
➜ gel plate is covered by buffer solution which conducts charge
Process of electrophoresis
➜ DNA samples are first digested using restriction enzymes to cut them at specific recognition sites (35 to 40℃)
➜ Tank is set up while restriction enzyme does its job
➜ Agarose gel is poured into a gel tray and left to solidify. A row of wells is created at one end and is it submerged in an electrolyte solution that conducts electricity
➜ Loading dye is added to tubes containing digested DNA and it is then loaded into wells using a micropipette
➜ once wells are loaded with diff samples, apply an electrical current
➜ negative electrode is connected to end of the plate with the wells as DNA frag will move towards anode
➜ DNA fragments move through fel at diff speeds and smaller frags move faster and therefore further in a time period
➜ At end of time period, buffer solution is poured away
➜ fragments transferred onto absorbent paper or nitrocellulose and then heated
➜ Probes are added
➜ probe adheres to the DNA and stains fragment
Probes
➜ single-stranded DNA sequences that are complementary to the VNTR regions
➜ radioactive label - makes x ray film go dark and forms dark bands
OR
➜ fluorescent stain / dye - shines when exposed to UV forming coloured bands
Pros of probes
➜ can be used to locate a specific gene needed for GM
➜ can be used to identify the same egne in a variety of diff genomes
➜ can be used to identify presence/absence of a specific allele that causes genetic disease
Microarrays
➜ scientists can place diff probes on fixed surface
➜ applying DNA under investigation to the surface can reveal the presence of mutated alleles that match fixed probes as sample DNA will anneal to complimentary fixed probes
➜ sample DNA must be first broken into smaller fragments and amplified via PCR
➜ DNA microarray can be made with fixed probes
Protein separation
➜ diff amino acids determine charge of proteins and charge depends on pH so buffer solutions used to keep pH constant
➜ Prepared by:
∘ denature to break disulfide bonds
∘ manipualte proteins into rod shapes
➜ Gel electrophoresis can be used to show genotypes of individuals by separating polypeptide chains produced by different alleles
e.g used for analysing haemoglobin proteins of sickle cell anaemia etc
Frederick Sanger
1970’s
➜ uses modified nucleotides called dideoxynucleotides that pair with nucleotides on template strand
➜ when DNA poly encounters dideoxynucleotides it stops replicating
dideoxynucleotides
➜ modified nucleotides
➜ prevent the next base from bonding to the template strand as it can’t form a phosphodiester bond with the dideoxynucleotide
e.g
A T C C G A T
T A G G٭
- Next base would be C and it can’t bind as the G٭ prevents it from binding to the G and the dideoxynucleotide itself
Procedure for chain termination sequencing
(sanger)
➜ 4 test tubes are prepared that contain DNA to be sequenced, DNA poly, DNA primers, free nucleotides and ONE of the dideoxynucleotide (A٭ C٭, T٭, or G٭ ) in ech of the 4
➜ radioactive probes is added to all
➜ test tubes incubated to a temp that allows DNA poly to function
➜ primer anneals to the start of the template producing a short section of doubel stranded DNA
➜ DNA poly attaches to double strand section and begins DNA replication using free nucleotides and form hydrogen bonds
➜ At any time the DNA poly can insert dideoxynucleotides instead by chance
➜ As each test tube only has one type of dideoxynucleotides we can work out the final nucleotide of each chain in every test tube
e.g f the test tube contains A٭, then researchers will know that the final nucleotide of every chain in that test tube is A
➜ As this is random the DNA chains are diff lengths
➜ after incubation the DNA chains (AKA developing strands) are separated from the template DNA
➜ let it sit and use gel electrophoresis to separate DNA fragments of diff lengths
➜ gell will have 4 wells for each A٭, C٭, T٭, and G٭
٭ = dideoxynucleotides
High-throughput sequencing
➜ diff method of fragment separation to sangers
➜ Each type of dideoxynucleotide is labelled using a specific fluorescent dye:
➜ Adenine base = green
➜ Thymine base = red
➜ Cytosine base = blye
➜ Guanine = yellow
➜ single stranded DNA is separated via capillary electrophoresis which has high resolution
◦ laser beam used to illuminate all of the dideoxynucleotides, and a detector then reads the colour and position of each fluorescence
◦ detector feeds the information into a computer where it is stored or printed out for analysis
Next-generation sequencing
&
Nanopore
➜ anything after sangers method is referred to as next generation sequencing (NGS)
➜ thousands to millions of DNA mols sequenced in parallel
➜ very fast
➜ Nanopore sequencing is currently being developed by scientists
DNA profiling (genetic fingerprinting)
enables scientists to identify crime suspects and identify corpses because every person (apart from identical twins) has repeating short non-coding regions of DNA (20 to 50 bases) that are unique to them, they are called variable number tandem repeats (VNTRs)
- VNTR’s are regions of human genome - they are where the bases are repeated
- they are located in non coding DNA (introns) they have a high mutation rate and are unique
- STR are short tandem repeats and smaller than VNTR as they degrade slower
