6.1.3 manipulating genomes Flashcards
what is a genome?
all of an organisms genes
what does DNA sequencing determine?
order of nucleotides in a DNA sample
what is the purpose of PCR?
to amplify DNA so you can find out the order of bases
why is PCR used?
- DNA profiling (forensics)
- finding cancerous mutations
- finding out what genes do (characterization)
describe DNA fragmentation
- before PCR, DNA cut into fragments
- palindromic sequences in DNA recognized by restriction endonucleases which cut/ digest the DNA at these sites
- leaving “sticky ends” that overhang
- each restriction enzyme is specific to a sequence and comp to its active site
state the 4 stages of PCR
- denaturation
- annealing
- elongation
- amplification
describe stage 1 (denaturation) of PCR
- DNA heated to 94-6 degrees
- 15-20 secs
- DNA separates into single strands
describe stage 2 (annealing) of PCR
- cool mixture to 68
- primers anneal to DNA by comp base pairing
describe stage 3 (elongation) of PCR
- heat to 72
- let DNA (taq) polymerase to extend primers
- copies of target DNA synthesized using target strands as templates
why does the genome need to be fragmented before sequencing?
- genome is too big
- smaller fragments have fewer errors
- more accurate
- otherwise would take too long
compare PCR and in vivo gene cloning
- PCR - quicker, takes few hours
in vivo- weeks - PCR - less equipment needed - tube and heat
in vivo- multiple tubes and agar plates - PCR- easier and less costly (set to run and left)
in vivo - have to pick out and transfer the colonies - PCR - combines selection of DNA and amplification (primer selects only target gene to be copied)
in vivo- needs probe to correctly identify the gene
5 stages of DNA profiling
- extraction of DNA
- digesting sample
- electrophoresis
- hybridization
- evidence
explain how the locations of the fluorescent on DNA reveal which genes are now active
- mRNA binds to gene probes by comp base pairing
- the more active the gene the more mRNA produced
- during transcription
- so more fluorescent indicates more mRNA
stages of genetic engineering
- the gene of interest is cut out from the DNA using restriction enzymes
- the gene is inserted into a plasmid (vector) using DNA ligase and into bacteria
- bacteria that contain new DNA are called transgenic bacteria
- they are grown on agar plates containing antibiotics so you can distinguish them from bacteria that have not taken up any new DNA
- a fluorescent probe can be used to identify bacteria that have desired sequence of DNA
explain how the enzyme DNA ligase is used
- used to join pieces of DNA together to make recombinant DNA (DNA that is joined together from different organisms)
- reseals cut ends of DNA
- forms the sugar phosphate backbone
what is reverse transcriptase enzyme?
RNA made into DNA (reverse of transcription)
outline the procedure by which bacteria can be genetically modified to produce renin.
- cut out renin gene using restriction enzyme
- if you don’t know the gene sequence - extract mRNA and use reverse transcriptase to make ss cDNA and use DNA polymerase to make it ds
- cut the bacterial plasmid with restriction enzymes, leaves sticky ends
- cut the rennin gene with same restriction enzyme leaving comp sticky ends
- use DNA ligase enzyme to anneal and join sugar phosphate backbones to form recombinant DNA
- insert the vector into the cell using heat shock (with CaCl2) or electroporation
- Transformation used to identify when cells have taken up vector containing new gene
suggest 2 ways which bacteria that take up modified plasmid can be identified.
- fluorescent marker ( marker gene) bacteria that fluoresce under UV light have plasmid
- insert antibiotic resistant genes into new genes
- grow on agar containing antibiotics
- survivors will have the modified plasmid
describe somatic gene therapy
- replacing mutant gene with healthy allele
- not passed onto offspring
- involves liposomes ( artificial vesicles that package the DNA and fuse with the cell membrane to deliver DNA to target cells)
- normal body cells
describe germ line gene therapy
- inserting healthy allele into germ allele
- usually egg
- can be sperm or fertilized egg
- differentiation and growth occurs
- added allele present in every cell of body
- passed onto offspring via gametes
what is gene therapy?
treating generic disease by changing natural genotype
describe a difference between somatic and germ line gene therapy
somatic targets specific tissues in need of treatments germ line doesn’t
suggest 3 developments that have led to an increase in the speed at which DNA can be sequenced
- high throughput sequencing
- shotgun sequencing
- pyrosequencing
differences between the base G (in DNA)
and an ATP molecule
G base contains guanine
ATP contains adenine
G has 1 phosphate
ATP has 3 phosphates
G contains deoxyribose
ATP contains ribose
In G phosphate it attached to C3
In ATP no phosphate attached to C3
how does DNA sequencing allow the sequence of amino acids in a polypeptide to be predicted?
- used to determine order of bases
- order of bases codes for the sequence of amino acids
- each triplet of bases code for 1 amino acid
a virus has a rapid mutation rate so scientists worked to develop a vaccine against it. outline how DNA sequencing and bioinformatics are used to increase the effectiveness of a vaccination programme against the virus.
DNA sequencing
- find the order of bases
- virus has a high mutation rate which leads to many STRAINS being produced
- scientists can predict the viral strain
- so the vaccine contains the correct antigen
Bioinformatics
- uses computer software to analyze store and organize biological data about DNA, RNA and proteins.
- the data is universal and shared internationally
- it can identify the source of the outbreak and vulnerable populations
- so the vaccination programme can target certain areas
how can bioinformatics be useful in determining whether a newly sequenced allele causes a genetic disease?
- the data base can be used to find the known base sequence of the healthy normal allele
- computational analysis allows fast comparison with the newly sequenced allele
- the a.a sequence in held in the data base
- computer modeling of new protein structure from data base
explain why only selected sections of non-coding DNA are used during DNA profiling in humans
- in most humans the genome is similar / most genes are the same
- using coding DNA would not provide unique profiles
- parts of non-coding DNA contain short tandem repeats (STRs)
how do you calculate how many DNA fragments might be present after a certain number of cycles?
- 2^n
- 2 because it is double stranded
- n = number of cycles
how many DNA fragments might be present after 12 cycles of PCR. 1 DNA fragment at start. represent answer as log10 value.
- 2^12 = 4096
- log(4096) = 3.61
- answer is 10^3.61
why might the number of DNA fragments calculated after 12 cycles not be achieved in practice?
- temperature damages fragment
- template strands could rejoin after being separated rather than primers annealing
- lack of primers
- primers fail to anneal to fragment
name of enzyme used in PCR to synthesise new DNA strands
taq polymerase
what is the purpose of the buffer/ alkaline solution in gel electrophoresis?
solution carries charge/ current to separate fragments
why is taq polymerase used instead of dna polymerase?
- thermostable
- does not denature at 95 degrees when the strands are separated
- so PCR can be cycled repeatedly without stopping to reload the enzyme.
describe how DNA can be visualized after electrophoresis.
fluorescent tags which fluoresce under UV light
explain how the gene is inserted into a vector
- plasmid cut by restriction enzyme
- leaves sticky ends
- gene cut with same restriction enzyme
- leaves comp sticky ends
- so gene is inserted into vector by comp base pairing
- using DNA ligase
how do you obtain the gene of interest if you don’t already know the gene sequence?
- make single stranded complementary DNA
- using enzyme reverse transcriptase
- use DNA polymerase to make double stranded DNA
4 differences between somatic and germ line gene therapy
- somatic - genes not inherited / passed onto offspring
germ line - genes inherited - somatic - gene introduced into body
germ line - gene introduced into egg/ sperm cell - somatic - only some cells get functional gene
germ line - all cells get functional gene - somatic - short term
germ line - long term
ethical concern of testing on animals
arguments for and against animal testing
for -
1. animal closely related to human
2. helpful to human health
3. both primates
against -
1. can cause physical harm
2. animal has no choice
3. animal suffers
