6.3 - Manipulating Genomes Flashcards
DNA Sequencing def
A technique that allows genes to be isolated and read
Cloning DNA mechanism - using bacteria
- Gene to be sequenced is isolated
- Using restriction enzymes, from a bacterium
- DNA inserted into a bacterial plasmid - a Vector
- Inserted into a bacterium host (E. coli)
- When cultured, divided many times,
- Enables plasmid with DNA inserted to be copied many times
PCR Def - Polymerase Chain Reaction
- A method of artificially amplifying DNA to get many copies of the same sample
- Used to make enough DNA to test multiple times (crimes, genetic profiling etc.)
DNA Primers def
- 10-20 bases of single stranded DNA
- Complementary to base pairs of strand of DNA you are trying to replicate
Uses of DNA primers
- Used for DNA sequencing
- And for PCR to bind to section of DNA so (Taq) DNA polymerase can bind
- (Taq) DNA Polymerase cannot bind to single stranded DNA
PCR key temps for each step
Denaturation - 95 Degrees C
Annealing - 68 Degrees C
Elongation - 72 Degrees C
Denaturation of DNA def
When double-stranded DNA splits into single strands
What does PCR rely on?
- DNA is made of two anti parallel backbone strands
- Each strand of DNA has a 5’ end and a 3’ end
- DNA grows only from the end
Base pairs pair up according to complementary base pairs according to base pairing rules - A, T and G, C
Annealing (DNA) def
When a primer joins with complementary base pairs
- occurs at 68 Degrees C
How does PCR differ from DNA replication
- PCR - heat to 95C to separate complementary strands
- DNA replication - DNA helicase separates complementary strands
- PCR - DNA Primers needed for polymerase to join and reaction to begin
- DNA replication - no DNA primers needed for complementary base pairing to occur
- PCR - does not copy whole chromosomes
- DNA replication - copies whole chromosomes
- PCR - repeats immediately after one cycle done
- DNA replication - repeats once every cell cycle
- PCR - Taq DNA polymerase used
- DNA Replication - DNA polymerase used
- PCR - artificial DNA required
- DNA Replication - Natural DNA replication
- PCR - uses Mg2+ coenzymes
- DNA replication - no coenzymes required
What is Taq DNA polymerase
- Isolated from Taq DNA
- Obtained from Thermophilius Aquaticus
- A thermophilic extremophile bacteria (survives in harsh/hot conditions)
- Stable at high temperatures
PCR simple steps
- Small fragments of DNA to be copied is mixed with DNA nucleotides
- Mixed with primers
- Mixed with Taq DNA polymerase
- Mixed with Mg2+ cofactors (for polymerase
- Heated to 95 Degrees C
- Temp cooled to 68 C - Annealing
- Temp increased to 72 C - Elongation
- Process is repeated over and over again
Applications of PCR
- Tissue Typing
- Detection of oncogenes - specific gene cancer is found - tailored medication
- Detecting mutations
- Identifying viral infections
- Monitoring spread of infectious diseases
- Forensic science - DNA profiling, crime scenes, etc
- Research - analyse ancient base sequences/genomes
Similarities between DNA replication and PCR
Both use polymerase enzymes to catalyse formation of phosphodiester bonds between sugar phosphate backbone
Both utilise free nucleotides to form double stranded DNA from sing,e stranded DNA
Both polymerase enzymes form phosphodiester bonds in 5’ to 3’ direction in double stranded DNA
State the function of PCR
Mass replication of DNA strands
- amplifying DNA strands
Why are high temperatures used during … stage - at 95C?
To denature the enzymes present in Taq DNA
Why is temperature increased during … stage - 72C
Optimum temperature for Taq DNA polymerase enzymes to function
- Catalyse formation of phosphodiester bonds between complementary base pairs
What is electrophoresis?
What is it used for?
- A method of separating and ordering DNA fragments or proteins based on size
- used so that the fragments can be identified and analysed
- used in sequencing and DNA profiling
What charge does DNA have?
- Where does it move to in electrophoresis?
DNA is slightly negatively charged
- so moves towards the cathode
Electrophoresis of DNA mechanism
- Small amounts of DNA can be amplified using PCR
- DNA is cut into smaller fragments using restriction enzymes. (The same restriction enzyme must be used to cut the fragments from any of the individuals involved in the identification for forensics)
- The fragments are placed into the wells at the end of the gel plate where the negative electrode (cathode) will be
- The plate is immersed into a tank filled with buffer solution and an electric current is passed through the tank (1‐2 hours)
- DNA is negatively charged (due to the phosphoryl groups of the sugar‐phosphate backbone) and so are attracted to the other end of the plate, where the positive electrode (anode) is, so the molecules diffuse along the gel to the other end
- The shorter fragments move further in the same period of time than the longer ones
- The banding pattern is invisible so the DNA must be stained with ethidium bromide and then viewed under UV light to observe the final banding pattern
Electrophoresis of proteins
- done in the same way as DNA electrophoresis
• Sodium dodecyl sulfate (SDS) is added the proteins to give them equal negative charge
• This means that they can be separated by molecular mass (rather than charge)
Uses of protein electrophoresis
Analyse proteins by mass in order to diagnose medical conditions, e.g.
- sickle cell anaemia
- diseases in which patients have higher level of fetal haemoglobin than they should
DNA Profiling info
(AKA Genetic Fingerprint)
- a way of identifying individuals by characteristics of their DNA
- this is used to compare compare the DNA of more than one individual
- most human DNA is the same - so most would not be suitable for comparisons
- so hair/saliva is used to obtain cheek cells and DNA in nucleus
Short Tandem Repeats (STR) info
- STR are loci on the genome composed of 2-10 base pairs which repeat 5-50 times in a row
- the number of times the STR repeats varies at each loci which varies from person to person
- we can use these to compare the DNA of different individuals
What is a DNA probe?
A short single-stranded length of DNA that is complementary to a section of the DNA being investigated
How could the probe be labelled?
- A radioactive marker (usually with P32) in one of the phosphate groups in the probe strand
- once the probe has annealed (bound) by complementary base pairing to the piece of DNA, it can be revealed by exposure to photographic film
- a fluorescent marker that emits a colour in exposure to UV light.
- these may also be used in automated DNA sequencing
Why are DNA probes useful?
- to locate a specific gene needed for use in genetic engineering
- to identify the same gene in a variety of different genomes from different species when conducting genome comparison studies
- to identify the presence of a specific allele for a particular genetic disease - or a susceptibility to a particular condition
Chances of one person having the same STR as another person
- from 13 different STR loci:
- 10^13
- almost impossible
How do we find out the number of STRs a person has at each location?
Electrophoresis - more STRs - larger DNA fragments- moves less far in electrophoresis
How to create a DNA profile mechanism
- DNA obtained from all people to be compared, e.g. via mouth swab to obtain cheek cells/ saliva, hair etc.
- DNA amplified using PCR
- DNA from all people cut into different size fragments using the same restriction enzyme
- DNA from different people will be different sizes because the number of STRs will differ vary between each person - DNA fragments are separated based on size using gel electrophoresis
- people to be compared are loaded into different wells - DNA fragments examined (small fragments move further on agarose gel plate)
- DNA banding patterns from each person compared
What is used for DNA profiling?
- Using different polymorphic STR sequence variation in different people to compare
- STR - Short Tandem Repeats
How are STR sequences separated?
Gel electrophoresis
Uses of DNA profiling
- Maternity/paternity testing
- Forensic science, identify victims from body parts, etc.
- Analysis of disease - type of haemoglobin to diagnose anaemia/sickle cell anaemia, Huntingdon’s, etc.
- Studying evolutionary relationships - find common ancestors between species - the more similar the DNA banding patterns, the more closely related
DNA profiling def
AKA - genetic fingerprinting
- a method used to produce a specific pattern of DNA bands from an individual’s genome
What does DNA profiling rely on?
Relies on short, repeating sequences of DNA that are found within the non-coding regions (introns) of DNA
How to extract ancient DNA
DNA from old bones
What are restriction enzymes/endonucleases?
- what are they used for?
A type of enzyme that cut up DNA at a specific sequence of bases called recognition sites
- used to cut the DNA into fragments that leave STRs intact - must not cut STRs
How to separate DNA fragments
Using gel electrophoresis
Which fragments travel quicker in gel electrophoresis?
Smaller fragments travel quicker and therefore move further along the gel
Interpreting two different DNA profile samples which are the same
Suggests that they were taken from the same person
Interpret DNA profile from two different samples that have around half DNA bands in common
Suggests a close genetic relationship between the two people
- as alleles or genes have been shared or inherited between those two people
Why are the same restriction enzymes used during electrophoresis
So they cut the DNA of each different sample at the same identification points/loci in the DNA samples
DNA Sequencing def (school)
The process of working out the order of bases on a DNA molecule/gene
Outline the roles of PCR in sequencing a genome.
2 Marks
- to amplify/make copies
- with a range of different lengths
Outline the role of electrophoresis in sequencing a genome.
2 Marks
- to put fragments of DNA is size/length order
- to read base sequences/orders of bases
Outline the role of digestion of DNA by restriction enzymes in sequencing a genome.
(2 Marks)
- cuts the genome into smaller fragments
- can cut vectors/plasmids (for gene library)
Suggest why a genome has to be fragmented before sequencing.
2 Marks
- genome is too large to sequence
- accuracy is better/fewer errors with smaller fragments
- can divide the DNA over different labs to study individual segments/fragments - at the same time
What is the purpose of PCR in investigating the genetic fingerprinting of deceased persons?
- PCR amplifies DNA - increases the amount of DNA available to be used in fingerprinting
- So enough DNA is available for genetic fingerprinting
What is the advantage of the enzyme used in PCR being thermostable?
(2 Marks)
- can withstand high temperatures
- would not be denatured at these higher temps.
Describe how genetic fingerprinting is carried out.
6 Marks
DNA extracted from sample
DNA hydrolysed/cut into segments using restriction endonuclease enzymes
Must keep core sequences intact
DNA fragments are separated using electrophoresis
(details about electrophoresis:)
put DNA micture into wells
pass an electric current through agarose gel
immerse the gel in alkaline solution - separate two DNA strands
Radioactive marker/probe is added to better view the DNA fragments
Areas with probe identified using X-ray film, etc.
What are DNA primers?
1 Mark
Short lengths/fragments of DNA nucleotides
- single stranded DNA
Why are DNA primers added during PCR?
1 Mark
bind to the single-stranded DNA to form double stranded DNA
allows binding/attaching of Taq DNA polymerase at that end
prevents two DNA strands re-joining
What is meant by a gene?
2 Marks
A length of DNA
that codes for a specific protein
Suggest why two different primers may be needed during PCR.
1 Mark
- sequences at end of target sequence are different
- as DNA strands are antiparallel, DNA primers act at the 3’ end so the primers would have to be different at each end of the strands
Explain why a particular primer will only bind to the DNA of one species.
(2 Marks)
kjkjb
how to work out number of DNA fragments produced after x no. of repeats of PCR
2^n x no. of DNA fragments at the start
What is a DNA ladder?
hsfehslf
Genetic Fingerprinting mechanism
- retrieve tissue from subject - e.g. saliva to obtain cheek cell
- fractionate the cells
- ultra-centrifugation - isolate and obtain the DNA/genetic material
- Restriction enzymes used to cut the chromosomes into smaller pieces/DNA fragments
- PCR used to amplify the DNA - obtain more DNA fragment replicates to be used in electrophoresis
- Pass charge to separate the DNA fragments out - shorter fragments travel further
- DNA ladder used to see how long the DNA fragments are
- use a DNA probe to visualise the DNA fragments more easily - fluorescent dye, use UV light
Genetic Engineering - Recombinant DNA Mechanism
- Go to the Beta cells and extract the mRNA
- mRNA will mainly be INsulin mRNA (and no introns)
- Reverse transcriptase is used to convert mRNA to duplex DNA
- Use cell fractionation to isolate plasmids from bacteria
- cut plasmids open with a restriction endonuclease
- This makes staggered cuts
- Also generates single strand overhangs: called sticky ends
- Add a promoter region and a marker gene (such as for fluorescent protein) to the Insulin gene
- as well as sticky ends complimentary to the plasmid
10 . Mix plasmid and insluin fragment together - sticky ends will attract. - Use DNA LIgase to seal the phosphodiester bond at the sticky ends
- So that the inserted gene cannot fall out again
- The recombinant plasmid must now be inserted into a host bacterium
- either:
- Using a viral vector such as a bacteriophage
- via heat shock - to destabilise and cause pores in the membrane, together with calcium ions
- ultrasound to punch holes in the membrane
- electroporation - The bacteria is now cultured to identify which have taken up the recombinant plasmid
- Via streak plating - you can pick out individual colonies expressing the marker gene
Why is bacteria used to make insulin as opposed to pig insulin extractions?
- no ethical issues
- easier to manufacture
- cheaper to manufacture
- ## bacteria produce human insulin - less chance of allergic reactions
During PCR, why do we cool the mixture down RAPIDLY from 95 Degrees during denaturation to 68 for annealing?
Allows the primers to join with complementary base pairs before DNA strands snap and join back together again.
- favours primers joining the single strands of DNA first
Cut plasmids and lengths of foreign DNA can join. What features of their ends allows them to join?
(2 Marks)
- base pairs on foreign DNA are complementary to base pairs on single-stranded DNA
- so hydrogen bonds can form between them
- as sticky ends would form at ends of the strands
Explain why base pairs are a suitable way of measuring the length of DNA.
(2 Marks)
- DNA made of base pairs
- Each base pairs is the same length
Aims of human genome project
- work out the order of sequences of all three billion base pairs in the human genome
- to identify all the genes in the human body
Benefits of human genome project
- improved genetic testing
- location of genes that might be linked to increased chances of inheriting a disease
- new gene therapy
- new knowledge of how humans have evolved
- personalised medicines
- synthetic biology
Comparing genomes between species uses (via human genome project)
- evolutionary relationships can be explored by conipsidering similarities of genomes between two species.
- beneficial genes are conserved by evolution
- identify genes which have been altered to give rise to differences between organisms
- identification of genes common to most/all living things
- can give clues to relative importance of these genes to life
- differences in gene interactions leading to different proteins being produced
- can be researched
- medical research can be carried out
- compare genome of pathogenic and non-pathogenic bacteria
- to identify genes responsible for causing disease
Which molecules/proteins are used to compare genomes between species
Cytochrome C
Hameoglobin
(DNA)
Reasons to compare genomes between individuals
- investigate the relationship between which genotypes cause which phenotypes
- early human migration can be mapped by comparing genomes of humans from around the world
- medical advances can be made by possibly producing drigs specific to an individuals genome, to maximise its effect
- epigenetics - help our understanding of diseases, like cancer, that may or may not develop in gentically similar organisms
How could a genome sequence allow us to predict the strcurure of a protein?
(2 Marks)
Three bases = codon
Codon codes for one amino acid - same codon codes for same amino acid (DNA is universal)
Chain of amino acids = primary structure of protein
This relies on scientists knowing the location of introns and exons
Synthetic biology
Jsjsjjs
How to acquire a specific gene - mechanism
- mRNA removed from cells expressing the gene and used as a template
- regerse transcriptase forms complementary DNA strand, primers,
- DNA polymerase turns this into a double stranded length of DNA coding for original protein
- automated polynucleotide synthesiser builds gene (if nucleotide sequence known)
- PCR primer used to amplify gene (if sequence known)
- DNA probe used to locate gene which is cut out using restriction enzymes
Restriction enzymes info
From bacteria and archea to protect from attacks by phage viruses
Cut up foreign viral DNA
Own DNA is protected as is methylated at recognition site (named according to where they are from, e.g. E.coli)
- Mg ions may be needed as a cofactor
Can be blunt or sticky
What does DNA ligase do?
Catalyses condensation reaction to join the pentose sugar and phosphate groups of the DNA backbone to form a phosphodiester bond
What is recombinant DNA?
A bacteria that has a gene from another species
How to insert a gene into a plasmid - mechanism
Cut open plasmid using same resriction enzymes (same ones isolated gene with)
This leaves complementary sticky ends
Sticky ends of gene and plasmid anneal and H bonds form between bases
Why is the same restriction enzyme used when cutting out the gene, and inserting it into a plasmid
Has the same sticky ends
4 different ways to DNA across plasma membrane (for genetic engineering)
- outline them
Heat shock treatment - bacteria alternated between 0-42C with CaCl, membrane becomes more porous
Electroporation - a high voltage pulse applied to cell to disrupt membrane
Electrofusion - electricsl fields help introduce DNA into cells
Transfection - DNA packaged into a bacteriophage, which can then transfect the whole cell
Recombinant plasmid - T plasmids are inserted into the bacterium into plant cells, and infect plant cells
Golden bullet - small pieces of gold (inert) with DNA, are shot into the cell
Q)
Answer:
