Molecular Genetics Flashcards
What does genetic variation result in
Different phenotypic characteristics
How does genetic difference exist
Between individuals within or between different populations
What is the scale of variation
From gross alterations in the human karotype to single nucleotide changes
What are the 2 types of variations
. Polymorphisms
. Disease causing mutations
How much DNA do we share with each other
99.9%
What does genetic variation lead to
Evolution
Species evolved due to mutations in genetic code
Most mutations do not have an effect on us - lead to phenotypic differences - genetic variation - slightly different
What are the types of mutations
. Point mutations
. Frameshift mutations
Point mutations
Single nucleotide base is changed, inserted or deleted from a sequence of DNA or RNA due to a simple mistake during DNA replication in meiosis
Result in often little consequence, sometimes severe consequences
e.g. sickle cell anaemia
Frameshift mutations
Caused by a deletion or insertion or inversion in a DNA sequence that shifts the way the sequence is read
Such mutations are more dangerous and change the codon
What can some mutations lead to
Disease
Non - functional proteins = damaging phenotype - no evolutionary benefit = disease causing mutations
Alter reading of codons in transcription/translation = protein chain at end is significantly different
What type of mutation has a more serious consequence
Frameshift mutations
What percentage of the population have Polymorphism
> 1%
Very abundant
What is Polymorphism unlikely to be causative of
Genetic disease
It causes variation in normal phenotype, sometimes but not always
What is the consequence of polymorphisms?
Can affect disease predispoistion, progression or drug response, when combined with other genetic and environmental factors ( disease risk factor )
Why are polymorphisms unlikely to be caused by genetic disease
- As you wouldnt find in such abundance
- Evolution took disease causing mutations out of the gene pool, if it was disease causing, it would lead to evolutionary deficit = taken out of gene pool
- Affect progression of diseases - risk factors - association with specific diseases and affect how you respond to different drug combinations = influence over diseases but most of them cause natural variation
What are the 4 common types of polymorphism?
- SNPs- single nucleotide polymorphism - point mutations
- Indels - small insertion/deletions
- Large scale copy number polymorphism - CNPs or CNVs
- Tandem repeats
What are tandem repeats
Larger regions of nucleotide material that can be repeated throughout genetic code
A sequence of nucleotide can be repeated numerous times in sequence
what is the most common type of polymorphism?
SNPs
make up 80% of the 0.1% difference between individuals
What is SNP
- Point mutation
- Single nucleotide substitution of nucleotide with another
Where can SNPs occur
Anywhere on genome in DNA strand 1. Within gene . Coding ( exons ) . Non-coding ( introns) 2. Between genes . Non-coding ( intragenic region )
Where is it more frequent for SNPs to occur and why
Within non-coding region due to selection pressure
Less likely to cause abnormal functioning protein if not active in region of DNA strand that codes for protein itself
During RNA splicing, non - coding regions do play role in transcription factor binding and RNA splicing of specific genes - influence over proteins
Do SNPs change phenotype
- Most SNPs are neutral with no phenotype change
- 3-5% have a functional role and do not change phenotype - variation by v.small amount
Types of SNPs
. Synonymous - codes for the same amino acid - by chance it could be a mutation which changes it for another nucleotide which by chance codes for same a acid when read as triplet = NO EFFECT
. Non-synonymous - alters the poly peptide chain
which has 2 possibilities
1. Missense mutation - alters amino acid sequence
2. Nonsense - results in premature stop codon - resulting in a shorter polypeptide chain = non functional protein
What may SNPs in non coding regions do
May alter gene splicing/transcription factor binding/regulation
What are Indels
Insertion or deletions of nucleotide sequences of 2-10,000 base pairs
What is 2nd most common polymorphism
Indels
What type of mutation occurs in indels?
Frameshift mutation
2 - 10,000 base pairs
What are the 2 types of tandem repeats
- VNTRs - Variable Number Tandem Repeats
aka mini-satellites - 10-100 base long segments repeated - STRs ( Short tandrem repeats )
aka micro-satellite ( smaller ) - 2-9 base long segments repeated ( fragment lengths )
Where are Tandem repeats found
Throughout genetic code but have little consequence
Non coding regions
Use of tandem repeats
DNA fingerprinting - match it up
What are CNPs
Entire gene duplicated or deleted within genetic code
Larger number of nucleotides repeated
How is a polymorphism different from a mutation leading to a specific disease ?
- It is due to the prevalence in population
-If mutation is found in <1% of population = mutation
-If mutation leads to loss of function or aberrant levels of normal protein or mutant protein its a disease causing mutations
THESE ARE NOT REFERRED TO AS POLYMORPHISMS
Where do disease mutations often occur
In exons
What happens if disease mutations occur in exons
Cause protein insufficiency/protein overexpression = disease - undesirable phenotype = can cause death
What are disease mutation referred to as ?
. Point mutation
. Frameshift mutation - insertion/deletion
. Inversions - nucleotide sequence flipped around
. Copy number variations
mini and micro satellites are rare in exons
What is non-disjunction?
- During meiosis1 when one daughter cell has an extra chromosome and the other daughter cell has less
- At end meiosis2 the same daughter cell will not contain a particular chromosome , while others have an extra copy
- Chromosomes don’t separate properly - lead to wjole chromosome genetic diseases
- Fertilisation using a gamete with an extra chromosome will result in a zygote with 3 copies of that chromosome ( trisomy )
What is the most common trisomy
Down’s syndrome
What is Down’s syndrome
Where a person has 3 copies of chromosome 21 instead of 2
What are examples of other autosomes displaying trisomy
. Edward’s disease - 3 copies of chromosome 18
. Patau’s disease - 3 copies of chromosome 13
Effect of non-disjunction in sex chromosomes?
. Turner’s syndrome - females with only one x chromosome - no Y or 2nd X chromosome - Monosomy chromosome
. Kleinfelter’s syndrome - XXY males - additional C chromosome as a male
. Metafemale syndrome - XXX
What is effects of Turner syndrome
Short, shoulder attached onto higher up on neck
Ears slightly down and during devlopment they move up = webbing of neck
What is structure of chromosome ( nomenclature )
Has 2 arms - a short arm “p” ( petite ) and long arm “q” separated by a centromere.
Differential banding patterns - different density along chromosome
How can you determine what someones genetic mutation is
Only using molecular biology techniques
What is symptoms of Down Syndrome
- Moon face
- Holes in heart
- Mental retardation
What can we use to look at the structure and number of chromosomes ?
Karyogram - reffered to as the karyotype = look at chromosomal changes
What can you compare in a karyotype
- Chromosome count- right number of pairs - not trisomy/monosomy
- Centromere position - any changes in length of arms
- Banding pattern - any differences on chromosome?
.-Sex chromosomes - M or F
Process of karyotype
- Take cell sample from blood
. Culture nucleated cells in dish
. Stop cells dividing ( arrest ) during METAPHASE ( chromosomes condense here )
. Extract chromosome from a single nucleus in sample
. Stain with giemsa ( as you cant see - too small and transparent ) - gives optical density - under high power microscope you can see individual chromosomes
What is DNA probe ?
- A single stranded DNA chain that contains a nucleotide sequence specific for the gene or chromosomal region of interest.
- Detect nucleotide chains containing the complementary sequence ( complementary to region of genetic code interested in )
- Highly specific - even if 18 bps long
- Creates synthetic polynucleotide chain
- Simplest
What are the two types of DNA probes
Oligonucleotide probe ( aka primers ) - short - 18 - 20 nucleotides long Polynucleotide probe - v long ( 1000s base pairs )
In FISH what are DNA probes labelled with and why
A fluorescent tag
Fluorescent protein or radioactive materal
Look where DNA probe is binding to genetic code
What is FISH
Fluorescence In Situ Hybridisation
A molecular technique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity.
What happens in FISH
. Fluorescent DNA probes can be applied directly to chromosomes.
. So the number of chromosomes per nuclei can be counted
SEE LECTURE SLIDE FOR EXAMPLE
What are restriction enzymes ?
- Enzymes which cleave single stranded DNA at certain nucleotide sequence/specific sites along gentic code into restriction fragments
- Initiates cleavage - breaking between bases on both sides of DNA strand - snaps single DNA strand into 2 pieces
.- Enzymes bind to specific sequence of nucleotide in genetic material
e.g. Hae 111 - specifically binds to GGCC sequence by recognising 4 bp sequence and binds to DNA at that specific point - Thye break apart bonds between nucleotides in DNA
Where do restriction enzymes come from
Bacteria
- Isolate from different strains of bacteria – part of a bacteria’s immune system.
- Open to attack by viruses, viruses contain their own genetic material and if a virus infects a bacteria it has a batch of these different enzymes within it which then it releases to target DNA
e. g. Escherichia coli, haemophillus Influenzae
Target sequences of restriction enzymes
Short ( 4 - 8 nucleotide pairs ) - bind to multiple points along DNA strand
Where to restriction endonucleases cut
For any given DNA molecule, one specific restriction enzyme always cut at the same sequence sites
What is the difference between blunt ends and sticky ends ?
. restriction enzymes don’t always cut at the same sites
. blunt end- enzyme cuts DNA strand through the middle
. sticky end- enzymes don’t cut straight through DNA strand = overalapping regions
Uses of restriction enzymes
- Probe for known mutations - know sequence around a mutation and expect to be there = use the enzyme to chop DNA strand
- Mutations can abolish or create restriction sites
- Digestion generates a series of restriction fragments which are formed by cutting DNA strand into multiple different fragments
What is gel electrophoresis
Process used to separate DNA and RNA fragments based upon their size
Process of gel electrophoresis
. Both DNA and RNA are negatively charged due to phosphate group
. They are run through a density gel
. Apply current through gel
. Get banding pattern
. Use a ladder of known fragment sizes in one lane to be able to determine your DNA or RNA sizes - one lane to run sample that contains lots of different gragments of specific known base pair lengths
Which fragments travel faster in gel electrophoresis and why
Shorter fragments due to less resistance
Because if you want nucleotides - chains to flow through something which has cross linked strands in it, theres resistance
Gel of gel electrophoresis
Use carb jelly e.g. agerose - extract from seaweed - lots of different molecules interlocked together
Which direction do fragments travel in gel electrophoresis
Downwards
Top = cathode = negative
Bottom = anode = positive
Negative ions on nucleotide fragments from phosphate are repelled from cathode and attracted towards positive anode = DNA fragments run downards through gel
How can you visualize the DNA after gel electrophoresis ?
The gel is soaked in a dye ( ethidium bromide ) which binds to DNA and fluoresces under UV light
What is PCR ( polymerase chain reaction ) used for ?
- To determine genetic variation between DNA samples
- Particular DNA sequence can be copied accurately and rapidly for analysis
. Can amplify target sequence 100 million fold in 2-3 hrs - Generate sufficient quantities of target DNA sequence for analysis
- Genetic testing
- Specific PCR amplifications to check for genetic mutations
What is the main ingredient used in PCR ?
. TaqDNA polymerase
What does PCR use DNA polymerase for
to copy DNA template using repeated cycles of replication
Functions well at body temp ( 37.5C) = useless - want to break the bonds - so want to inactivate DNA polymerase
What is TaqDNA polymerase derived from
Thermophilic bacterium - Thermus Aquaticus
Loves high temps - higher temps than humans
Found around acquatic volcanic events
What does the polymerase do
Guided to the target sequence using short synthetic oligonucleotide ( primer ) which hybridise to the desired DNA sequence.
Primer sequence is complementary to the DNA
Process of PCR
- Heat double stranded DNA ( 94-96) degrees to separate the 2 DNA strands = denatured DNA
- Slight cooling of reaction mixture ( 50-65 ) degrees to allow the hybridization/annealing of primers to single stranded DNA ( oligonucleotide)
- Extension ( by reading and recruiting free floating nucleotides )heat to 72 deg and TaqDNA polymerase activates and starts to read genetic material = forms a complimentary copy of the DNA strand
What happens in the first 3 cycles of PCR ?
. 1st cycle - two double stranded DNA molecule
. 2nd cycle - four double stranded DNA molecule
. 3rd cycle - eight double stranded DNA molecule
and so on many cycles happen
Goes through multiple cycles = end up with a lot of DNA
After 30/40 cycles you exponentially increase amount of DNA you have
What are the application of PCR ?
. Provides numerous DNA templates for mutation screening
. Forensic application
. mRNA templates can be analysed
. Gene specific PCR reactions can be designed which discriminate between DNA sequence differing by a single nucleotide (SNPs)
. Amplify sequences from minute quantities of target DNA
what is reverse transcription ?
RT-PCR can be used to check if mRNA is being transcribed and if protein can be translated - analyse mRNA templates
. reverse transcribe mRNA into cDNA using reverse transcriptase
- Same process but different enzyme
- Add primer to mRNA
- Chilled - primer anneal to RNA
- Add reverse transcriptase = cDNA
Ingredients of PCR
- TaqDNA polymerase
- Primers - floating around - original binding box - binding site for TaqDNA polymerase
- Free floating nucleotides - complementary to what irs reading on DNA
DNA sequencing examples
Malaria Parasite 2008 Human November 2003 Drosophila Melanogaster 2003 Mouse 2002 Escherichia coli January 2001 Saccharomyces cerevisiae 1996
What does chain length termination use
ddNTP - Di - Deoxy - nucleotide - Triphosphate
Process of chain length termination
. Addition of ddNTP
.ddNTP lacks the 3’ -OH group and cannot form a phosphodiester bond/ sugar phosphate backbone as you extend nucleotide sequence to form polynucleotide = cant bind any more nucleotides = chain termination
. Requires incorporation of fluorescent labelled phosphate
.Lots of PCR reaction occurs
. 4 possible variants of nucleotide ( A,C,G,T)= 4 different coloured tags = know what nucleotide is bound at that point
. Run through agerose gel, tiny capillary tube, short ones travel faster.
. Laser shining = detects which fluorescent tag is going through it
. At the end you have different length fragments with different fluorescent tag on them
. Laser+ tag identify colour tag to see which fluorescent tag goes with each nucleotide
What are some bio methods used to understand molecular genetics ?
. Allele-specific PCR - detecting point mutations
. Restriction fragment polymorphism analysis - detecting point mutation
. Detecting variable number tandem repeats
What is Allele specific PCR
. Each sample is tested with three primers in PCR reaction instead of two. 1 common primer, other 2 are specific for each of those different alleles
. One primer is the same for both reactions the other exits in two different versions
.One primer is specific for normal sequence
. One primer is specific for the mutant sequence
. If product TT = homozygous
. If one C allele and one T allele = heterozygous
. If one nucleotide difference between these = not bind to each other
.If have 2 specific alleles interested in and known theres a point mutation between the 2 - wnat tosee if samples have: Allele 1, Allele 2 or both alleles
SEE LECTURE SLIDE FOR EXAMPLE
What is restriction fragment polymorphism ?
. Restriction enzyme cut/cleave DNA samples. Run gel electrophoresis and see how large
. SNPs at the site that restriction enzyme cleaves will prevent binding and subsequent cutting
. Therefore you can distinguish if a person is homozygous (1 band) , heterozygous (2 bands) or not affected by a mutation
. This method only works if you know specifically what mutation you’re looking for and probing to see if someone has it or not
SEE LECTURE SLIDE FOR EXAMPLE
Detecting VNTRS two methods
- PCR
- Restriction enzyme cleavage
These 2 methods are useful if the mutation is known or region is suspected. But to identify new mutations you need to do DNA sequencing
Detecting VNTRS
Use PCR or restriction enzyme to create DNA fragments to run on a gel electrophoresis
See how the variable number of tandem repeats- the more there is the bigger the fragment.
Detect larger variables - sequence of nucleotides can be repeated many times in a sequence
Repetitive region of nucleotide sequence. Often duplicate region of 10 - 100 bases
SEE LECTURE SLIDE FOR EXAMPLE
Are mutations always bad
NO
they are the heart of evolution and natural selection - no mutation = look the same
they can lead to better prognosis in disease
e.g. MDR1 Gene Polymorphisms Affect Therapy Outcome in Acute Myeloid Leukemia Patients
e.g. C3435T SNP associated with GOOD cancer therapy outcome
