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A-Level Biology > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

1
Q

Genome

A

Entire genetic material of an organism - DNA in the nucleus and the mitochondria combined

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2
Q

How is DNA layout?

A

Chromosomes made up of hundreds of millions of DNA base pairs - genes only make up about 2% of your total DNA - these are exons

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3
Q

What are introns?

A

Large non-coding regions of DNA that are removed from messenger RNA before it is translated into a polypeptide chain

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4
Q

DNA profiling?

A

Producing an image of the patterns in the DNA of an individual - assists in the identification of individuals/family relationships

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5
Q

What is DNA profiling referred to as?

A

Genetic fingerprinting since every individual’s DNA profile is unique

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6
Q

5 main stages in producing DNA Profile

A

Extracting the DNA
Digesting the sample
Separating the DNA fragments
Hybridisation
Seeing the evidence

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7
Q

Extracting the DNA

A

Using Polymerase Chain Reaction - tiniest fragment of tissue can give scientists enough DNA to develop a profile

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8
Q

Digesting the sample

A

Strands of DNA are cut into small fragments using special enzymes called restriction endonucleases - different enzymes cut DNA at a specific nucleotide sequence known as a restriction site

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9
Q

How many cuts do restriction endonucleases make?

A

Two - one through each strand of DNA

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10
Q

Why do scientists use restriction endonucleases?

A

Give scientists ability to cut the DNA strands at defined points in the introns ; use a mixture of restriction enzymes that leave the repeating units or satellites intact so the fragments at the end of process include a mixture of intact mini and micro satellite regions

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11
Q

Separating the DNA fragments?

A

Using electrophoresis - the way charged particles move through a gel medium under the influence of an electric current - gel is immersed in alkali in order to separate the DNA double strands into single strands - single stranded DNA fragments are then transferred onto a membrane by southern blotting

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12
Q

Hybridisation

A

Radioactive or fluorescent DNA probes are now added in excess to DNA fragments on the membrane ; these are short DNA/RNA sequences complementary to a known DNA sequence which bind to complementary strands under particular conditions of pH and temperature

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13
Q

What does hybridisation do?

A

Identifying micro satellite regions that are more varied than larger mini satellite regions

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14
Q

Seeing the evidence

A

If radioactive labels were added to the DNA probes - X-ray images could be taken of the paper/membrane ; or fluorescent tags under UV light - fragments give a pattern of bars which is unique except for identical siblings

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15
Q

What is PCR used for?

A

Amplifying a DNA sample

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16
Q

Ingredients for PCR

A

Excess of the 4 nucleotide bases (in the form of deoxynucleoside triphosphates)
Small primer DNA sequences
Enzyme DNA polymerase
ALL IN A THERMAL CYCLED

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17
Q

Step 1 of PCR

A

Temperature is increase to 90/95 which denatures the DNA by breaking hydrogen bonds holding them together so they separate

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18
Q

Step 2 PCR

A

Annealing of the primers - temperature is decreased to 55/60 and the primers bind (anneal) to the ends of the DNA strands - they are needed for the replication of the strands to occur

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19
Q

Synthesis of DNA PCR

A

Step 3

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20
Q

Step 3 PCR

A

Temp is increased to 75 for optimum temperature for DNA polymerase
DNA polymerase adds bases to the primer building up complementary strands of DNA and so producing double stranded DNA identical to the original sequence

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21
Q

What enzyme is used in synthesis of DNA?

A

Taq polymerase - obtained from thermophilic bacteria found in hot springs

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22
Q

Uses of DNA profiling

A

Forensics - dna profiles from blood, semen etc is compared to a criminal DNA database
Prove paternity of a child - demonstrates evolutionary relationships between different species
Identifying individuals who are more at risk from developing certain diseases - certain non-coding micro satellites have been found to be associated with increased incidence of disease

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23
Q

DNA profiling summary

A

Used with DNA sequencing to make more confident risk assessments
Producing an image of the patterns in non-coding DNA of an individual

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24
Q

DNA sequencing

A

Determine the precise order of nucleotides within a DNA molecule

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25
First sequencing technique?
Sanger sequencing enabled him to read 500-800 bases at one time - first genome they sequenced was phiX174 - virus that attacks bacteria
26
Refinement of DNA sequencing technique?
Swapping of radioactive labels for coloured fluorescent tags which led to scaling up and automation of the process - led to capillary sequencing version which is still used today
27
HGP
Massive international project established in 1990 to map the entire human genome - making the data freely available to scientists all over the world
28
Why was human genome first draft complete so quickly
Automation of sequencing techniques and development of faster computers meant that project was ready two years ahead of schedule and under budget
29
What are terminator bases?
Modified versions of the 4 nucleotide bases which stop DNA synthesis when they are included - A terminator will stop DNA synthesis at the location that an A base would be added etc etc ; the terminator bases are also given coloured fluorescent tags Terminator bases lack an oxygen on third carbon
30
A
Green
31
G
Yellow
32
T
Red
33
C
Blue
34
What does capillary method start with?
DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases
35
Step 2 dna sequencing
Mixture is placed in thermal cycles and rapidly changes temperature - 96 degrees would cause double stranded DNA to separate and at 50 degrees the primers anneal to the DNA strand
36
Step 3 dna sequencing
At 60 degrees, DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template
37
Step 4 dna sequencing
Each time a terminator base is incorporated - synthesis of DNA is terminated ; as chain terminating based are present in lower amounts and are added at random, it results in many DNA fragments at random lengths - after many cycles, all the possible DNA chains will be produced with the reaction stopped at every base ; they are separated according to length by capillary sequencing (fluorescent markers are used to identify the final base on each fragment) - lasers detect different colours and thus order of the sequence
38
Order of bases in capillary tubes
Shows the sequence of the new complementary strand of DNA = used to build up original strand ; data fed into computer that reassembles the genomes by comparing fragments and finding the areas of overlap between them - these genomes can then be researched
39
Next-generation sequencing
DNA sequencing technologies have become faster and more automated - new, automated, high-throughput sequencing processes - instead of using gel of capillaries, the sequencing reaction takes place on a plastic slide known as a flow cell
40
How does a flow cell work?
Millions of fragments of DNA are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments - as all of the clusters are being sequenced and imaged at the same time, technique is known as ‘massively parallel sequencing’
41
How refined are these DNA sequencing processes?
Extremely efficient - 3 billion base pairs of human genome can be sequenced in days and those of a bacterium in less than 24 hours ; high-throughput sequencing also means cost has fallen so more genomes can be sequenced and they open up a range of questions that scientists can ask on the genome in many new ways
42
Bioinformatics
Development if software and computing tools needed to organise and analyse raw data - stats tests, mathematical models and algorithms etc (make sense of a lot of data)
43
Computational biology
Build theoretical models of biological systems - 3D structures of proteins from sequencing Determine evolutionary links/genes linked to diseases
44
Genomics
Analysing structure and function of genomes
45
Analysing pathogen genomes
Find sources of an infection Identify antibiotic resistant strains of bacteria Track of diseases like Ebola Identify genetic markers in pathogens for use in vaccines
46
Species genome analysis
DNA barcoding - common sections that vary between species Mitochondrial dna from cytochrome c - cellular respiration enzyme Have nit come up with regions for fungi and bacteria (downside)
47
Phylogeny
Basic mutation rate - can be calculated - how long ago two species diverged
48
Proteomics
Amino acid sequencing
49
Spliceosomes
Exons joined together by enzyme complexes called spliceosomes ; in a variety of ways so many proteins from one mRNA
50
Synthetic biology
Ability to sequence genomes and understand how it translates to amino acids - store and manipulate data
51
Synthetic biology examples
Genetic engineering Fixed/immobilised enzymes New genes replacing faulty genes - development of cystic fibrosis (functional genes replace faulty ones) Synthesis of entire new organism (artificial genome)
52
Transgenic
Organism that carries a gene from another organism
53
Isolate desired gene
Restriction endonucleases cut gene from DNA at specific base sequence Some are blunt Others are sticky - one strand longer than other strand ; unpaired/exposed bases makes it easier to insert desired gene into DNA of another organism
54
How else to isolate gene?
Using reverse transcriptase to produce a single strand of complementary dna - makes it easier to identify the desired gene as some cells make a lot of mRNA
55
Vectors
Bacterial plasmids - contain marker gene so for example they may have been engineered to have a gene for antibiotic resistance Bacteria have taken up the plasmid by growing in media containing antibiotic - bacteria will still grow
56
How to cut open plasmid
Same restriction endonuclease - plasmid has complementary sticky ends - DNA ligase phosphodiester bonds
57
If DNA fragment inserted successfully
Marker gene will not function - often fluorescence or colour change ; HAS BEEN ENGINEERED SUCCESSFULLY IF IT DOES NOT FLUORESCE
58
Transferring the vector
Plasmid with recombinant DNA is transferred to host cell in TRANSFORMATION
59
First way of transformation
Culture in a calcium rich solution and increase temperature - membrane is permeable and plasmids can enter
60
Second way of transformation
Electroporation small electric current is applied to the bacteria - makes them porous and plasmids move in POWER HAS TO BE CONTROLLED else membrane will be permanently damaged
61
Electro fusion
Tiny electric currents are applied to membranes of two different cells - fuses cells and nuclear membranes together to form a polyploid cell continaining both DNA Used to create a hybridoma
62
How many different restriction endonuclease needed
2 for each cut
63
Recombinant plasmid
Genes from organism + marker genes - ONLY FOR DNA Transgenic is for organism
64
Restriction endonucleases
At restriction sites
65
Reverse transcriptase
Allow mRNA to be transcribed - extracted and mix with RE Converts mRNA back into COMPLEMENTARY DNA AND PUT INTO PLASMID Useful because it’s mature MRNA (already spliced) SAVES A LOT OF TIME - DO NOT NEED TO PROCESS INTRONS IN PLASMID - SAVES ENERGY FROM BACTERIA
66
Where do restriction endonucleases cut in vector
At marker gene Blue marker gene recombinant - DOES NOT PRODUCE BLUE PIGMENT BECAUSE CANNOT BE TRANSCRIBED Second marker gene - antibiotic resistant gene (can survive with antibiotics)
67
What enzyme joins fragments together to make recombinant DNA?
DNA ligase
68
Plasmids only transformed into
Bacteriaaaaa
69
Blue colonies or
White colonies
70
Somatic cell gene therapy
Replacing mutant allele with a healthy allele in the affected somatic body cells - a temporary solution as healthy allele is passed on every time a cell divides by mitosis but somatic cells only have limited lives so replaced by stem cells with faulty allele STILL PASSES ON FAULTY ALLELE TO ANY CHILDREN
71
Germ line cell gene therapy
Healthy allele into the germ cells of an embryo after fertilisation - individual born healthy and pass onto offspring
72
Downside of her line cell gene therapy
Potential impact on an individual is unknown Human rights of unborn individual is violated without consent and irrevocable Enables people to choose desirable or cosmetic characteristics ETHICAL concerns
73
Pharming
Production of human medicines - creating animal models or human proteins
74
Animal models
Genes deleted so they are more likely to develop cancer - develops certain diseases
75
Human proteins
Human gene coding for a medically required protein - genetic material of a fertilised cow - fertilised transgenic female is then returned to mother ; when baby matures and gives birth produces milk with proteins to be harvested
76
Ethical issues
Welfare of animals (in pharming for example) Human genes in animals Making animals commodities
77
Insect resistance
Inserted gene into soya beans to produce bt protein that is toxic to many pests - no need for pesticides (or weed killer sometimes)
78
Benefits of GM crops
Increase yield - no pesticide spraying helps environment Resistant to diseases Herbicides can reduce competition Enhanced levels of vitamins and grown in wider conditions all year round Produce human vaccines
79
GM crops cons
Non-pest insects may be damaged by toxins in GM plants May produce supersedes ; transferred genes in wild populations - reduces biodiversity People may be allergic Reduce commercial value and demand - LONG SHELF LIFE
80
Patent
Legal patent means less economically developed countries cannot use them - flood/drought resistant crops unable to afford the GM seed CANNOT SAVE THE SEED FOR GROWTH THE NEXT YEAR Share technological advancements without patent ✅

A-Level Biology (37 decks)

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