Section 3: Genetics Flashcards
1
Q
Gene
A
A discrete unit of heredity
Consists of a specific DNA or RNA sequence
2
Q
Allele
A
Alternative versions of a gene
3
Q
Homozygous
A
An organism having a pair of identical alleles for a trait, e.g. pp
4
Q
Heterozygous
A
An organism having two diff alleles for a trait, e.g. Pp
5
Q
Genotype
A
An organism’s genetic makeup
e.g. PP, Pp, pp
6
Q
Phenotype
A
An organism’s expressed traits
7
Q
When replicated, a chromosome has __ sister chromatids
A
2
8
Q
Karyotype
A
The visual representation of a species chromosomes arranged in pairs at mitotic metaphase
9
Q
Karyotype - replicated chromosomes
A
View replicated chromosomes because this is where we see chromosomes easily - when they’re condensed and replicated = easiest to characterise
10
Q
Reproduction
A
When an entity undergoes division and results in the production of 2 entities of the same kind
Based on reproduction of cells and the cells’ genetic material
11
Q
Reproduction - unicellular vs multicellular organisms
A
Unicellular - produces an entire individual
Multicellular - allows growth and repair
12
Q
Mitotic cell division results in…
A
Production of 2 genetically identically daughter cells
13
Q
Asexual reproduction
A
One parent
Single parent contributes all the genes
Clones (copies)
Mitosis
14
Q
Sexual reproduction
A
2 parents
Each parent contributes half their genes
Vary genetically
15
Q
How often are somatic cells reproducing
A
Continuously
16
Q
Mitosis and cell cycle
A
Interphase (~90%):
G1
S phase (DNA synthesis)
G2
Mitotic (M) phase:
Mitosis
Cytokinesis
17
Q
Amount of DNA cells during cell division
A
S phase: amount of DNA doubles because each chromosome replicates itself
G2: amount is maintained
M phase: reduces back down to half - production of 2 genetically identical cells
18
Q
Non-replicated chromosomes
A
Consist of one piece of double-stranded DNA
19
Q
Mitosis: S phase
A
Duplication of chromosome
Consists of 2 sister chromatids which are genetically identical
20
Q
Mitosis: Interphase (G2)
A
Chromosomes already replicated
Chromosomes in form of chromatin
21
Q
Chromatin
A
Uncondensed chromosomes
Chromosomes spend most of their lifecycle in this uncondensed phase
22
Q
Chromatin
A
Uncondensed chromosomes
Chromosomes spend most of their lifecycle in this uncondensed phase
23
Q
Mitosis: Prophase
A
Chromosomes start to condense into sister chromatids
Centromeres visible
Asters start to move to either pole
24
Q
Why do chromosomes condense into chromatids
A
So it’s easier for them to divide
25
Chromosomes: Centromeres
Primary constriction in chromosome
| Where spindles attach
26
Mitosis: Prometaphase
Spindles have attached to the kinetochores at the centromeres
Nuclear membrane breaking down
Chromosomes start to line up on metaphase plate
27
Mitosis: Metaphase plate
An imaginary line drawn through the cell
28
Mitosis: Metaphase
Replicated chromosomes line up along metaphase plate with each sister chromatid pointing to each pole
As they get pulled apart by spindles, each sister chromatid goes to each pole
29
Mitosis: Anaphase
Sister chromatids continue moving towards each pole and cells start to divide (including the cellular components)
30
Mitosis: Telophase and cytokinesis
Cleave furrow
Nucleolus forming
Nuclear envelop forming
31
Mitosis - stages
```
(Interphase)
Prophase
Prometaphase
Metaphase
Anaphase
Telophase and cytokinesis
```
32
Life cycle
Sequence of stages in an organism's reproductive history
| Conception to production of a new offspring
33
Fertilisation
The fusion of 2 haploid gamete nuclei to form diploid zygote nucleus
34
Haploid
A single set of chromosomes (n)
35
Diploid
The condition in which each autosome is represented twice (2n)
36
Meiosis
2 successive nuclear division in which a single diploid cell forms 4 haploid nuclei
37
Humans
Multicellular diploid organisms
| 2n = 46, i.e. 23 pairs of chromosomes
38
Humans - reproductive cells
Sperm and eggs
Contain half the genetic material the rest of our body does
Egg + sperm --> zygote (restores 2n) --> develops into individual
39
Meiosis - stages
```
(Interphase)
Prophase I
Metaphase I
Anaphase I
Telophase I and cytokinesis
```
Prophase II
Metaphase II
Anaphase II
Telophase II and cytokinesis
40
Meiosis: Interphase
Chromatin
| Already replicated
41
Meiosis: Prophase I
Condensed down into chromosomes
| When 2 homologous pairs that are replicated come tgt, non-sister chromatids can cross over, i.e. exchange DNA
42
Does crossing over occur in mitosis
Yes, but not exactly the same because cells are identical
43
Meiosis: Metaphase I
Chromosomes line up in their homologous pairs
| Orientated randomly on metaphase plate - important for generating variation
44
Meiosis: Anaphase I
Replicated chromosomes move to either side of pole
45
Meiosis II
Individual chromosomes line up along metaphase plate and each of the sister chromatids goes to either pole
Results in 4 haploid gametes containing half the genetic material we started with
46
Meiosis: Independent assortment - which stage does this occur
Metaphase I
47
Meiosis: Independent assortment
Homologous pairs align in a random orientation on metaphase plate
Each maternal and paternal homologue assorts independently of other pairs
48
Meiosis: Crossing over - which stage does this occur
Prophase I
49
Meiosis: Crossing over
The exchange of genetic material between non-sister chromatids
Produces recombinant chromosomes, i.e. chromosomes carrying genes derived from each parent
Shuffles the genes within the chromosomes
50
Meiosis: Crossing over in humans
1-3 crossover events occur per chromosome pair
51
Meiosis: What processes add to genetic variation
Independent assortment
Crossing over
Random fertilisation
52
Meiosis: Why is crossing over essential
Essential for cell division otherwise don't line up and divide properly
53
Meiosis I vs II
I: homologous pairs lined tgt, and one of the pairs goes to one pole and other goes to other pole
II: separated chromosomes line up and get separation of sister chromatids
54
Mendelian laws: First law
Principle of segregation
The 2 alleles segregate during gamete production
The diploid state is restored by fusion of gametes
55
Mendelian laws: Second law
Principle of independent assortment
When on diff chromosomes, each allele pair assorts independently of other allele pairs during gamete formation
i.e. one gene behaves independently of other gene, but ONLY if those 2 genes are located on diff chromosomes
56
Chromosome theory of inheritance - parallel
Parallel behaviour of Mendel's genes and behaviour of meiotic chromosomes
- pairing
- segregation
- independent assortment
57
Cystic fibrosis
NZ's most common lethal genetic disease
| Normal/unaffected allele is dominant
58
Incomplete / partial dominance
Dominant allele is not fully expressed in heterozygote
Heterozygote has an intermediate phenotype because only having one copy allows you to convert only half the precursor
e.g. cross red + white = pink
59
Co-dominance
Full expression of both alleles in the heterozygote
60
Co-dominance: ABO blood group
Diff alleles responsible for expressing a carbohydrate on surface of RBCs
A expresses A carbohydrate
B expresses B carbohydrate
O expresses none
61
Co-dominance: ABO blood group - antibodies
AB: bursts with A and B
AA and AO: bursts with A
BB and BO: bursts with B
OO: doesn't burst
62
Co-dominance: ABO blood group - carbohydrate and alleles
Carb A: I(A)
Carb B: I(B)
No carb: i
63
The gene encoding for the ABO blood group is an example of a gene with _____
Multiple alleles
64
Co-dominance: ABO blood group - phenotype and genotype
A: I(A)I(A) or I(A)i
B: I(B)I(B) or I(B)i
AB: I(A)I(B)
O: ii
65
Multiple alleles
Every individual has 2 alleles, but in the population there can be more alleles
66
DNA fingerprinting
Each individual has 2 alleles (of diff sizes), but there are many alleles in the pop (~3000)
67
No of genes per genome vs no of chromosomes
No of genes per genome far exceeds the no of chromosomes
| Therefore must be more than one gene per chromosome
68
How many genes does each chromosome contain
Hundreds or thousands
| except Y chromosome which has ~16 genes
69
Are most of our genes inherited in the Mendelian fashion
No because they're all located on the same chromosome --> tend to be inherited tgt
70
Linked genes
Genes that are located on the same chromosomes and tend to be inherited tgt
Exceptions to Mendelian inheritance
71
Test cross
Crossing a heterozygous and homozygous recessive
72
Test cross: +
+ is dominant form
73
Results of a testcross - possible progeny
```
Aa Bb (non-recombinant)
aa bb (non-recombinant)
Aa bb (recombinant)
aa Bb (recombinant)
```
74
Results of a testcross: Independent assortment
Aa Bb 25%
aa bb 25%
Aa bb 25%
aa Bb 25%
75
Results of a testcross: Complete linkage
Aa Bb 50%
| aa bb 50%
76
Results of a testcross: Linkage with some crossing over
Aa Bb and aa bb = more than 50%
| Aa bb aa Bb = less than 50%
77
Recombination frequency =
(No of recombinants /
| total no of offspring) x 100 = %
78
Mapping a chromosome using recombinant frequencies assumes...
That crossing over occurs randomly, then the frequency that crossing over occurs should be proportional to the distance between 2 genes
79
1% recombinant frequency = ?
1 map unit or 1 centimorgan (cM)
80
What can recombinant frequencies be used for
Build linear chromosome maps
81
Genetic map
The order that loci occur along a chromosome
82
Linked genes - parental vs recombinant genes
Outside genes can't combine --> parental type
| Adjacent can recombine --> recombinant - the further apart they are on the chromosome, the more recombinant there is
83
Total recombination frequency for 2 genes tgt is less than the sum of the genes separately - why
Double crossing over can occur --> turns back into parental type --> reduces recombination frequency
Tends to result in underestimation of big genetic distances
84
Sex-linked traits: Morgan's first mutant - experiment + results
P generation: Homozygous female (red eyes), male (mutant X chromosome and Y chromosome) (white eyes)
F1 generation: All offspring red eyes
F2 generation: typical Mendelian 3:1 ratio, but all white-eyed flies males
85
XY - hemizygous
Many genes located on the X chromosome don't have a homologue (corresponding allele) on the Y chromosome
86
Sex-linked traits: Morgan's first mutant - F2 white eye
Female is heterozygous with a mutant, male is recessive with a Y chromosome, so must be a mutant with Y --> male
87
Heterogametic sex
The sex that produces two kinds of gametes and determines sex of offspring
In humans = male
88
Homogametic sex
The sex that produces one kind of gamete
| In humans = female
89
Chromosomes and sexual phenotype: XO
Missing an X chromosome
Human: sterile female
Drosophilia: sterile male
90
Chromosomes and sexual phenotype: XX
Human: normal female
Drosophilia: normal female
91
Chromosomes and sexual phenotype: XXX
Human: Fertile female
Drosophilia: Sterile female
92
Chromosomes and sexual phenotype: XY
Human: Normal male
Drosophilia: Normal male
93
Chromosomes and sexual phenotype: XXY
Human: Sterile male
Drosophilia: Fertile female
94
Chromosomes and sexual phenotype: XYY
Human: Fertile male
Drosophilia: Fertile male
95
Chromosomes and sexual phenotype: Turner's syndrome
XO - sterile female
96
Chromosomes and sexual phenotype: Klinefelter's syndrome
XXY - sterile male
97
SRY - functions
Determines sex
Triggers testicular development
A transcription factor (DNA binding protein)
98
Chromosome - pseudoautosomal region
At either end of chromosome is a pseudoautosomal region - can pair up to each other and undergo crossing over
99
SRY - pseudoautosomal boundary
SRY located v close to pseudoautosomal boundary, so in some cases crossing over occurs and transfers Y gene --> males
100
Do males / females contain female / male genes
Yes; males are always repressing female genes and females always repressing male genes
101
Androgen receptor gene - mutation
Results in partial or complete inability of cells to respond to androgens
102
Androgen insensitivity syndrome (AIS)
Even though individual has testes and are producing androgen, their body doesn't recognise it --> develop back into females
Extreme conditions: born female, but at 12 or 13 y/o, half of them turn into males
103
X chromosome inactivation
In females, each embryonic cell randomly inactivates one of the two X chromosomes
Some genes can escape from inactivation
104
Barr body
Inactivated X chromosome
| Densely stained objects in nuclei of females
105
Barr body genes
Most Barr body genes aren't expressed
106
Barr bodies - stability
Stable through mitosis, but the X is reactivated in cells that give rise to ova
107
Males vs females - sets of genes
Females have 2 sets, males have 1
108
X inactivation - tortoiseshell cat
Females consist of a mosaic of 2 types of cells; those with active X derived from father and active X derived from mother
Get a random expression of black and orange fur across the body in females only
109
Are all genes located on nuclear chromosomes
No - some are located in organelles, e.g. mitochondria and chloroplasts contain small circular DNA molecules
110
Mitochondria genes - inheritance
Mostly maternal inheritance
| Don't show Mendelian inheritance
111
Mitochondria genes - function
Most are involved in ETC and ATP synthase
112
Mitochondrial diseases
Nervous system and muscles are most susceptible to energy deprivation, so are most affected
113
Organelle genes resemble...
A bacterial genome
114
Mitochondrial myopathy
Causes weakness, intolerance of exercise and muscle deterioration
115
Types of meiotic nondisjunction
Nondisjunction of homologous chromosomes in meiosis I
| Nondisjunction of sister chromatids in meiosis II
116
Meiotic junction - common?
Quite common
117
Meiotic nondisjunction: Meiosis I
Both homologous pairs go to same pole
| Daughter cells: 2 of them has one extra chromosome, other 2 have 1 less chormosome
118
Meiotic nondisjunction: Meiosis II
Separation of sister chromatids doesn't occur
| Daughter cells: 1 has one extra chromosome, 1 has one less chromosome, 2 are normal
119
Aneuploidy
Abnormal number of certain chromosomes
120
Trisomic
1 extra chromosome (3 copies)
121
Monosomic
1 less chromosome (1 copy)
122
Nullisomic
Lack both chromosomes (0 copies)
123
Polyploidy
More than 2 complete chromosome sets
| e.g. triploid (3n), tetraploid (4n)
124
Alterations to chromosome structure - severity
Animals cope much better with alterations to chromosome structure than with extra sets of chromosomes
125
Alterations to chromosome structure: Deletion
Removes a chromosomal segment
126
Alterations to chromosome structure: Duplication
Repeats a segment
| Our genome is full of this
127
Alterations to chromosome structure: Inversion
Reverses a segment
| Results in decreased gamete viability
128
Alterations to chromosome structure: Translocation
Moves a segment from one chromosome to another, non-homologous one
129
Alterations to chromosome structure: Reciprocal translocation
Most common type
| Non-homologous chromosomes exchange fragments
130
Alterations to chromosome structure: Non-reciprocal translocation
A chromosome transfers a fragment without receiving a fragment in return
131
Alterations to chromosome structure: Gene families are formed from...
Duplication
132
Human disorders due to chromosomal alterations: Down's syndrome
Karyotype shows trisomy 21, the most common cause
| Correlates with maternal age
133
Smallest chromosome
Chromosome 21
134
Nondisjunction of sex chromosomes produces a variety of...
Aneuploid conditions, most of which appear to upset genetic balance less than conditions involving autosomes
135
Why is genetic balance less affected in sex chromosomes than in autosomes
Y chromosome carries few genes
| Extra copies of X chromosome are inactivated as Barr bodies
136
Can males have Barr bodies
Yes, but few
137
Klinefelter's syndrome
XXY
Individuals have male sex organs, are sterile and have abnormally small testes
Extra X is inactivated, but some breast enlargement and other female characteristics are common
138
Turner's syndrome
XO
Phenotypically female but sterile as their sex organs don't mature
If provided with oestrogen replacement, can develop secondary sexual characteristics
139
Chronic myeloid leukemia (CML)
Reciprocal translocation
A result of translocation between chromosome 9 and 22
In cancer cells in nearly all patients contain a short chromosome 22 and abnormally long chromosome 9
140
What is the phenotype of an individual determined by
Interactions between alleles at the same time, and in many cases at a no of diff loci, as well as by the environment
141
Pleiotropy
A single gene having multiple phenotypic effects
142
Pleiotropy - cystic fibrosis
```
CFTR gene mutation
Results in defective/absent Cl- channels
Results in multiple traits affected:
- chronic bronchitis
- poor absorption of nutrients in intestine
- block pancreatic and bile ducts
```
143
Epistasis
When one gene/locus affects the phenotype of another gene
| e.g. recessive e genes may result in no pigment regardless of B or b gene
144
Continuous phenotypic variation is...
A complex trait
145
Continuous phenotypic variation: Human skin colour
Controlled by many genes
Evidence suggests at least 3 genes
Dark skin allele (A, B or C) is incompletely dominant to other alleles
146
Continuous phenotypic variation: Human skin colour - alleles
AABBCC very dark, aabbcc very light
| Alleles have additive effect, so AaBbCc and AABbcc same skin colour because both have 3 dominant alleles
147
Continuous phenotypic variation: Human skin colour - what can help smooth the curve
Environmental factors
148
Phenotypic range
Range of phenotypes that can be produced
149
When is the phenotypic range broadest
Broadest for polygenic traits
150
Is there a gene for everything
No - some traits occur above the genetic level
151
Norms of reaction
The environmental effect on the phenotype
152
Norms of reaction - ABO blood group
Norm of reaction has no breadth = given genotype always results in same phenotype
153
What does norm of reaction allow us to do
Gives us a way to quantify the response of the phenotype by the environment
i.e. fix genotype and vary environment, how much does the phenotype vary?
154
When are norm of reactions greatest
For polygenic traits
155
Genetic diseases can have a big...
Environmental component
156
Developmental noise
Random events in development leading to variation in phenotype
A slight difference in division in the beginning will result in a significant difference at end
157
Epigenetic landscape
Imagines the organism is a ball sitting at top of valley
Through development, ball rolls down the landscape
Environment can push ball in diff directions
Shape of landscape is determined by genes which could pull it to diff shapes
Small changes in genes in an area could change where the ball moves
Ball seesaws up and down valley, but eventually reaches end - phenotype
158
Germline mutations - effect on gene pool
New germline mutations immediately change the gene pool of a pop by creating a new allele
159
Mutations in coding regions - commonality
Relatively rare
| Reflects high fidelity of DNA replication
160
Mutations - random?
Mutations are random - they occur in any gene without regard to the benefit of that cell
161
Somatic vs germline mutations
Somatic: not transmitted to progeny
Germline: transmitted to some or all progeny
162
Where in a gene do most mutations occur
```
Certain regions (introns) are more prone to mutations because they don't affect the protein produced
Fewer exon mutations
Most exon variation doesn't change amino acid sequence (neutral)
```
163
Area of mutation within germline can result in...
Either a normal or mutant progeny
164
Most mutations are ____
Neutral - no phenotypic effect
165
Genetic disorder / hereditary disease
When a mutation has an adverse effect on phenotype / function
166
Sickle-cell disease - mutation
Caused by substitution of a single amino acid in haemoglobin protein of RBC
Amino acid Glu --> Val --> change in function
167
Sickle-cell disease - what happens
When O2 content of individual's blood is low, the sickle-cell haemoglobin protein aggregates into long fibrous chains --> deforms RBC (more rigid) --> difficult to move through BVs
168
Sickle-cell disease - symptoms
Physical weakness
Pain
Organ damage
Paralysis (blockage of small BVs)
169
Sickle-cell disease - haemoglobin protein
2 alpha and 2 beta genes
170
Sickle-cell disease - commonality
Most common inherited disorder among people of African descent
171
Sickle-cell disease - 1 vs 2 copies
Individuals with 2 copies of sickle-cell allele manifest the disease fully
Heterozygous inherit some of the symptoms - said to have the sickle-cell trait
172
Population
A group of individuals of the same species that live in the same area and interbreed
173
Gene pool
The sum of all alleles of all genes of all individuals in the pop
174
Hardy-Weinberg equilibrium
Allele and genotype frequencies in a pop will remain constant from one generation to the next
175
Hardy-Weinberg equation - allows calculation of...
The expected genotype frequencies given the observed allele frequencies
176
Hardy-Weinberg is a model of ______
Stability
177
Calculation of allele frequencies: No of alleles =
2 x no of individuals with that genotype
178
Calculation of allele frequencies: Dominant allele frequency
(no of DD individuals x 2) + (no of Dd individuals)
179
Calculation of allele frequencies: Recessive allele frequency
(no of dd individuals x 2) + (no of Dd individuals)
180
Hardy-Weinberg equations
p^2 + 2pq + q^2 = 1
p + q = 1
```
p = dominant
q = recessive
p^2 = frequency of RR
pq = frequency of Rr
q^2 = frequency of rr
```
181
Conditions for Hardy-Weinberg
```
No mutations
Mating occurs at random
No selection
Extremely large pop size
No gene flow
```
182
Conditions for Hardy-Weinberg: No mutations
The gene pool is modified if mutations changes alleles
183
Conditions for Hardy-Weinberg: Mating occurs at random
If individuals mate with a subset of the pop, genotype frequencies change
184
Conditions for Hardy-Weinberg: No selection
Differences in survival and reproduction of individuals with diff genotypes can alter allele frequencies
185
Conditions for Hardy-Weinberg: No gene flow
Movement of individuals in and out of a pop can alter allele frequencies
186
Non-random mating affects...
The way alleles combine to form genotypes and alters genotype frequency
187
Outbreeding
Promotes variability
188
Inbreeding
Results in loss of variation
Matings between second cousins or closer are more likely to produce homozygous genotypes for otherwise rare autosomal recessive genes
189
Cystic fibrosis - A child resulting from mating between first cousins has a ____ greater risk of being affected
~7-fold
190
Self-fertilisation removes....
Variation
191
Genetic drift
The change in allele frequencies as a result of change events
Overall affect is a loss of genetic variation
192
Genetic drift has major effects in _____ populations
Small
193
Genetic drift: Founder effect
When a small pop branches off from a larger one
| By chance, the small pop may not have all the alleles present in the larger pop
194
Genetic drift: Population bottlenecks
Occur when a pop is drastically reduced in numbers
| Chance effects can result in loss or fixation of alleles
195
Genetic drift: Fixation
Loss of other alleles
196
Gene flow
Movement of alleles into or out of a pop due to movement of individuals or their gametes
Tends to reduce differences
197
If gene flow is greater enough...
It can result in 2 pops combining into a single common gene pool
198
Recombinant DNA technology
Genes or DNA from 2 diff sources are combined in vitro in the same molecule
199
In vitro
In the lab / test tube
200
Restriction enzymes - how do they work
Cut DNA at specific sequences 4-8 bp in length
201
Restriction enzymes - bacteria
Isolated from bacteria where they protect bacteria from intruding DNA, e.g. phages
202
How many restriction enzymes
Hundreds; most of which have diff recognition sequences and cut the DNA at diff places (specific)
203
Restriction sites - symmetry
Most are symmetrical but cleave the DNA backbone in a staggered / asymmetrical way --> single-stranded ends called sticky ends
204
Recombinant DNA - what DNA is combined
Usually combine an organism's DNA (e.g. human or bird) with bacteria DNA
205
What is the sticky end useful for
Cloning DNA
206
Bacterial plasmid
A naturally occurring small circular piece of autonomous DNA but have been genetically modified to act as a vector
Can replicate inside a host bacterial cell
207
Vector
A piece of DNA that can carry another piece of DNA
| Used to insert pieces of DNA into bacteria
208
Mixing vector and foreign DNA
Mixing them in the appropriate conc causes them to H bond to each other
209
If the insert DNA is cut with the same restriction enzyme as the cloning vector...
They'll have complementary sticky ends --> can form H bonds
210
DNA ligase
Covalently links 2 pieces of DNA tgt
| Catalyses formation of covalent bonds that close sugar-phosphate backbone
211
Cloning genes and DNA ligase
Insert DNA is cut with same restriction as vector
| DNA ligase permanently joins the strands tgt (because H bonds aren't permanent)
212
Lac Z gene
Contains restriction site
| Has a promoter which when active produces enzyme beta-galactosidase
213
How can we help prevent genes rejoining to each other instead of joining to plasmids
By dephosphorylating the ends
| But this will always occur to a certain extent
214
Cloning a single gene into a plasmid - steps
Take recombinant molecules and bacteria and punch holes in outer membrane
Give them an electric shock --> drives plasmids into bacteria
Grow for a few generations for holes to repair
Take mixture of bacteria and put them on agar plates with ampicillin
Any bacteria that don't have bacteria can't grow
If DNA is present that disrupts gene --> non-functional --> white - called blue-white selection
215
Genomic library
A collection of clones that tgt contain an organism's entire genome
216
Genomic library: Shotgun cloning
Instead of focusing on a single gene, cloning starts with a mixture of fragments representing the entire genome
217
Bacterial artificial chromosomes (BACs)
Commonly used as cloning vectors
Similar mechanism to plasmids but come from diff source
Have larger inserts (100-300kb) so need less clones and can be stored as cells in a multi-well plate
218
Disadvantage of plasmids
Can only take a relatively small chunk of DNA
| ~2000-3000 bp
219
cDNA library
A collection of clones containing all gene sequences that are expressed in a particular tissue
220
What are cDNA libraries made from
mRNA molecules
| As a result, only contains genes that are being expressed at time of RNA extraction, i.e. tissue specific
221
cDNA library - complexity
Less complex than genomic libraries
222
cDNA don't contain...
Promoters
UTRs
Introns
223
What are cDNA libraries useful for
Studying genes responsible for specific functions
224
% of genome codes for what
1% of our genome codes a protein
| 20% of our genome codes for a known function
225
cDNA - disadvantage
Not all genes are expressed throughout life of an organism and not all genes are expressed in all tissues
226
cDNA library - steps
Extract mRNA from cells
Design primer with sequence TTTT
Reverse transcriptase: once it has the 3' end, it can make a DNA copy of the mRNA
Degrade mRNA
At other end, use primer on cap and do same thing, but use a normal polymerase instead --> double-stranded DNA --> clone back into DNA
227
mRNA - distinctive features
One end has poly-A tail, other end has cap sequence
228
cDNA library - primer
A short sequence of single-stranded DNA that can anneal to another single-stranded DNA or mRNA
229
DNA hybridisation
Allows us to sort through the thousands of clones and find the gene of interest
Uses a DNA probe
230
DNA hybridisation: DNA probe
A single-stranded labelled DNA fragment with similar sequence to a gene of interest - sticks
H bonds to the complementary sequence
Tagged with a radioactive or fluorescent label
231
Polymerase chain reaction (PCR)
A 3-step process that produces millions of copies of a targeted region of DNA
232
What is PCR based on
A heat stable DNA polymerase, which generates the second strand of DNA from a single-stranded template
233
DNA polymerase can only extend...
Existing double-stranded regions, therefore requires a primer
234
Reagents in a PCR reaction
Heat stable DNA polymerase
Deoxyribonucleotides (dNTPs)
2 primers (one for each strand)
235
What does a PCR do
Amplifies / makes copies of specific regions in genome
| These regions are between the two primers
236
PCR - steps
Take genomic DNA and design 2 primers to either side of the region to be amplified
Heat DNA (denaturation) --> 2 strands come apart
Cool DNA (annealing) --> primers attach
Heat DNA (extension) --> makes a copy on either strand
--> 2 copies
237
Gel electrophoresis
Separates macromolecules (DNA or protein) based on their rate of movement through gel in an electrical field - molecular sieve
238
Gel electrophoresis - what does rate of movement depend on
Size
Electrical charge
Other physical properties
239
Gel electrophoresis: DNA separation - what does it depend most on
Size / length of fragment
240
Gel electrophoresis: What determines how big the holes are in the sieve
Conc of agarose
241
Capillary DNA sequencing (dideoxy chain termination method)
Involves using one DNA strand to synthesise a nested set of complementary fragments
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Capillary DNA sequencing (dideoxy chain termination method) - random?
Synthesis is terminated randomly by addition of a fluorescently tagged ddNTP (dideoxy..) rather than a dNTP (deoxy...)
243
Capillary DNA sequencing (dideoxy chain termination method) - fluorescent tag
There's a diff fluorescent tag for each type of ddNTP (A, C, G, T)
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Capillary DNA sequencing (dideoxy chain termination method) - what happens to the resulting pop of molecules
They're separated by electrophoresis with the fluorescent labels detected for each fragment
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Capillary DNA sequencing (dideoxy chain termination method) - what does the colour of the fluorescent tag on each fragment indicate
The type of nucleotide at the 3' end
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Human genome project - three stages
Linkage mapping
Physical mapping
DNA sequencing
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Human genome project: Venter - shotgun sequencing
Skips the first two steps
| Take a genomic library and sequence all of it
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Genomics
The study of genes, their interactions and relationships within a species and between species
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Human genome project - physical map
Takes pieces of DNA from libraries and hybridises them to locate where they fit in the genome
250
Human genome project: Shotgun sequencing - steps
Cut DNA into overlapping fragments short enough for sequencing
Clone fragments in plasmids or vectors
Sequence each fragment
Order sequences into one overall sequence with computer software
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Human genome project: Shotgun sequencing - restriction enzymes
A range of them are used to cut the genome at diff places
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Human genome project: Shotgun sequencing - overlap
Only need ~20-30 bp overlap between fragments to be unique in a genome --> will join them tgt
253
Bioinformatics
The application of computational methods to the analysis of large biological data sets
254
Prokaryotes vs eukaryotes: Size of genomes
Prokaryotes: relatively small genomes
Eukaryotes: many more but huge variation
- no strong relationship between complexity of eukaryote and size of genome
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Prokaryotes vs eukaryotes: No of genes
Prokaryotes: relatively small no of genes
Eukaryotes: many more no of genes but huge variation
- no strong relationship between complexity of eukaryote and no of genes
256
Gene density
No of genes that exist per mega base (Mb)
257
Prokaryotes vs eukaryotes: Gene density
Prokaryotes: v efficient genomes - high gene density
Eukaryotes: lower gene density - lots of space within our genome (esp humans)
258
What do most bacterial genomes consist of
Genes for proteins, tRNA or rRNA, with the remaining consisting of non-transcribed regulatory regions, e.g. promoeters
259
In eukaryotes, the majority of DNA neither encodes... or ....
Protein, nor is transcribed into RNA of known function
260
Humans have ____ more non-coding DNA than bacteria
10,000x
261
Where are non-coding DNA regions found in eukaryotes
Some within genes (introns) but others aren't
262
Types of DNA in human genome: Exons
Coding part of genome
| Very small amount
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Types of DNA in human genome: Regulatory sequences
Once you can produce sufficient no of proteins, it's when they're turned on/off that impacts development of organism
264
Types of DNA in human genome: Introns
Many are transcribed but don't get translated
265
Intron shuffling
Shuffles which introns are cut out
266
Types of DNA in human genome: Unique non-coding DNA
Non-coding region represented only once in genome
267
What is DNA fingerprinting based on
Repeats of DNA sequences
268
Number and size of introns in genes
Variable
269
Function of introns
Not clear, but many are known to be transcribed
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Pseudogenes
Non-functional
271
Transposable elements - steps
Start with a retrotransposon
Synthesis of single-stranded RNA intermediate
Reverse transcriptase synthesises 1st DNA strand
DNA pol forms 2nd DNA strand --> mobile copy of retrotransposon
Inserted --> forms new copy of retrotransposon
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Transposable elements - eukaryotes
2 classes;
DNA transposons - cut and paste themselves
Retrotransposons - make an RNA intermediate
273
What are transposable elements
Mobile DNA sequences found in genome of all organisms (ubiquitous)
274
Transposable elements: Movement
Has certain sequences at end which means it can move into various locations in genome and insert itself
275
Transposable elements - abundant?
Abundant in many genomes
| e.g. make up ~44% of human DNA
276
Transposable elements - mutation
Can often cause mutations by direct insertion into genes
277
Transposable elements - can promote...
DNA rearrangements, e.g. chromosome deletions, inversions, duplications
278
Gene duplication can result from...
Misaligned recombination during meiosis (unequal crossing over)
279
Gene duplication - process
There's a transposable element on each non-sister chromatid
Since they're similar in sequence, they line up out of register
Crossing over - one of the genes are moved to other sister chromatid
Results in one sister chromatid with 2 copies and other has none
280
Gene families
Groups of genes similar in sequence
281
Gene families - functional?
Some remain functional whereas others become non-functional, i.e. pseudogenes
282
Most gene families arise through...
Duplication of an existing gene
283
Gene families - functions
DNA sequence of genes within family may change --> diff genes may come to have diff functions
284
Gene families - size
Some can have only a few genes, some can have many hundreds
285
Why have some genes evolved into tandem arrays
Because cells need large amounts of some gene products, e.g. protein
286
Often, the multigene families consist of..
Identical tandemly repeated DNA sequences
287
Tandem gene families - examples
Genes for histones
| rRNA - helps cells makes millions of ribosomes
288
What are tandem gene families
Where genes copies are right next to each other
289
Translocations in evolution: Human and chimpanzee
Human chromosome 2 is a large metacentric chromosome that resulted from fusion of two acrocentric chromosomes of apes (12 and 13)
Forms telomere-like and centromere-like sequences in human chromosome 2
290
Metacentric
Centromere is roughly in middle
291
FOXP2 gene: Mice
Wildtype - 2 normal copies
Heterozygous - reduced ability to vocalise
Homozygous - absence of vocalisation
292
FOXP2 gene: Humans and chimpanzees
Differ in expression of FOXP2 gene
| May explain why humans but not chimpanzees can speak
293
FOXP2 gene: Humans and Neanderthals
DNA sequence of gene is similar
| Suggests Neanderthals may have been capable of speech
294
Biotechnology
The manipulation of organisms or their component parts to make useful products
295
Recombinant DNA technology has launched a revolution in ___________
Biotechnology
296
Genetic engineering
The in vitro alteration of genetic material and the reintroduction of the altered DNA or RNA into a living organism
297
Main difference between genetic engineering and traditional biotechnology
Genetic engineering manipulates DNA or RNA in vitro
298
Genetic engineering vs traditional biotechnology: No of genes
Genetic engineering: precise choice of one or a few genes
| Selective breeding: new combinations of many genes
299
Genetic engineering vs traditional biotechnology: Type of genes
Genetic engineering: genes from any species
| Selective breeding: only genes from related species
300
Genetic engineering vs traditional biotechnology: Gene expression
Genetic engineering: control of gene expression
| Selective breeding: no control
301
Is genetic engineering a substitute for traditional methods
No, but it's a complementary technology
302
Traditional biotechnology: Plant breeders - methods
Selective breeding
Cloning
Mutagenesis
303
Traditional biotechnology: Mutagenesis
Typically involves using either chemicals or radiation to induce mutations and thus enhance genetic variation
304
Traditional biotechnology: Agriculture
Variation in chromosome number has been exploited to create new plant cultivars
305
Cavendish banana
A sterile triploid
306
Plants - ease of manipulation
Easier to manipulate since they don't separate their somatic and germ cells
307
Plants - polyploidy
Can become polyploidy easily
| Often a strong correlation with the size of cells and therefore the size of fruit
308
Genetically engineered food - glyphosate
The active chemical in herbicides that inhibits amino acid synthesis
309
Initial vs recent concerns regarding genetically modified plants revolved around...
Safety
Public health
Environmental effects
Recent: involves branding of country as 'genetically engineered-free' and protecting this image
310
Personalised medicine
Where each person's genetic profile can inform them about diseases and conditions for which they may be at risk
311
Personalised medicine: Many human genetic disorders can now be diagnosed using...
PCR and hybridisation / sequencing
312
Personalised medicine: Sequence-specific primers
Used to amplify a DNA fragment, which is characterised/sequenced to identify the disease-causing mutation
313
Personalised medicine: What might it lead to
Where each individual's genome is sequenced at birth
314
Genetic test for sickle-cell disease
1. Blood sample from patient
2. Genomic DNA from WBCs
3. Region of β-globin gene amplified by PCR
4. Hybridised with probe
5. DNA spotted onto binding filters and hybridised with probe
315
Next-generation sequencing: How has it revolutionised DNA sequencing
By sequencing multiple strands of DNA simultaneously
| Sequences entire genome
316
Next-generation sequencing - steps
1. Genomic DNA is fragmented
2. Each fragment is isolated with a bead
3. Using PCR, 10^6 copies of each fragment are made, each attached to magnetic bead by 5' end
4. Bead is placed into a well with DNA polymerases and primers
5. Solutions of each of the 4 nucleotides are added to all wells sequentially then washed off
Repeat
317
Next-generation sequencing: A chemical technique is used to identify..
Which of the four nucleotides is added
318
CRISPR-Cas9 - function
Can make targeted deletions, insertions and single nucleotide changes in genomes
319
CRISPR-Cas9: What does it act tgt with
A 'guide RNA' made from the CRISPR region of bacteria
320
CRISPR-Cas9: What does it cut
Any DNA that is exactly complementary to the guide RNA
| Cuts both strands --> triggers DNA repair system
321
CRISPR-Cas9: What happens when there's no undamaged DNA for repair to use as a template
Repair system sometimes inserts or removes (random) nucleotides
If cut is directed at a gene, the repair often alters the gene so it no longer functions - 'knock out'
322
CRISPR-Cas9: Guide RNA
Directs protein to exactly the place to be cut
323
CRISPR-Cas9 - formation of Cas9-guide RNA complex
Active sites in Cas9 protein can cut DNA
Guide RNA engineered to 'guide' Cas9 protein to target gene
Together forms Cas9-guide RNA complex
324
CRISPR-Cas9: The guide RNA can be engineered to be complementary to...
A a target gene
325
CRISPR-Cas9: Repairing genes
CRISPR-Cas9 can be used to repair a gene, if copies of the functional gene are introduced with the system
326
Gene therapy
The introduction of genes into an afflicted individual for therapeutic purposes
327
Gene therapy - aim
To insert a normal allele of the defective gene into somatic cells of affected tissue
328
For gene therapy to be permanent...
The cells that have the normal allele must be cells that multiply throughout the patient's life
329
Gene therapy: Prime candidates
Bone marrow cells that include stem cells that give rise to all cells of blood and immune system
330
Gene therapy - steps
1. Insert RNA version of normal allele into retrovirus
2. Let virus infect bone marrow cells that have been removed from patient and cultured
3. Viral DNA carrying normal allele inserts into chromosome
4. Inject engineered cells into patient
