molecular genetics Flashcards
why do we all have different phenotypic characteristics ?
due to variation which is genetic difference between individuals .
what are the two types of variation ?
. polymorphisms
. disease causing mutations
what are the two types of mutation ?
. point mutations
. frameshift mutations
what is point mutation?
single nucleotide base is changed from a sequence of DNA or RNA due to a simple mistake during DNA replication in meiosis
what are the consequence of point mutation?
often little consequence
e.g. sickle cell
what is frameshift mutation?
caused by a deletion or insertion or inversion in a DNA sequence that shifts the way the sequence is read
what is the consequence of frameshift mutation?
. more serious
what percentage of population have polymorphisms ?
> 1%
unlikely to be cause of disease as it is abundent
what is the consequence of polymorphisms?
can affect disease progression or drug response
what are the 4 common types of polymorphism?
- SNPs- single nucleotide polymorphism
- Indels - small insertion/deletions
- large scale copy number polymorphism - CNPs
- tandem repeats
what is the percentage of identical DNA between humans ?
99.9%
what is the most common type of polymorphism?
SNPs
make up 80% of the 0.1% difference between individuals
what type of mutation occurs in SNPs?
. point mutation
single nucleotide substitution of nucleotide with another
where does SNPs occur?
1. within gene . coding ( exons ) . non-coding ( introns) 2. between genes . non-coding ( intragenic region ) SNPs in non coding region may alter gene splicing/transcription factor binding/regulation
where is it more frequent for SNPs to occur ?
within non-coding region due to selection pressure
what is property of SNPs?
most SNPs are neutral with no phenotype change
3-5% have a functional role and do not change phenotype
what are the types of SNPs?
. synonymous - codes for the same amino acid
. 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
what are indels?
. indels are insertion or deletions of nucleotide sequences of 2-10,000 base pairs
. second most common polymorphism
what type of mutation occurs in indels?
frameshift mutation
what is tandem repeats ?
. a sequence of nucleotide can be repeated numerous times in sequence
what are the two types of tandem repeats ?
- VNTRs - aka mini-satellites
10-100 base long segments repeated - STRs - aka micro-satellite
2-9 base long segments repeated
what are CNPs?
entire gene repeated or deleted
how is a polymorphism different from a mutation leading to a specific disease ?
it is due to the prevalence in population
mutation is found in <1%
if mutation leads to loss of function or expression of protein its a disease causing mutations
where do disease mutations often occur?
exons
what are disease mutation referred to as ?
. point mutation . frameshift mutation . inversions . copy number variations mini and micro satellites are rare in exons
what is non-disjunction?
. results in 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
what are types of diseases that can occur due to non-disjunction?
. down’s syndrome - 3 copies of chromosome 21
. 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
. kleinfelter’s syndrome - XXY males
. metafemale syndrome - XXX
describe chromosome shape ? ( nomenclature )
Each human chromosome has a short arm “p” and long arm “q” separated by a centromere.
what can we use to look at the structure and number of chromosomes ?
. karyogram which is then referred to as the karyotype
what is karyotype and why is it used?
the process to look at chromosomal changes cab be used to compare . chromosome count . centromere position . banding pattern . sex chromosomes
describe the process of karyotype?
. take cell sample . culture in dish . stop cell division during METAPHASE . extract chromosome from a single nucleus . stain with giemsa
what are examples of molecular biology techniques used in lab to look at mutation ?
. DNA probe
. restriction enzymes
. gel electrophoresis
what is DNA probe ?
A DNA probe is a single stranded DNA chain that contains a nucleotide sequence specific for the gene or chromosomal region of interest.
. DNA probe bind to the gene of interest
. in FISH DNA probes are directly labelled with a fluorescent tag
what is FISH ?
(FISH) is 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
what are restriction enzymes ?
. restriction nucleases are enzymes which cleave single stranded DNA at certain nucleotide sequence into restriction fragments
. enzymes bind to specific sequence of nucleotide
e.g. Hae 111 binds to GGCC
what do restriction nucleases derive from ?
bacteria
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
what is gel electrophoresis ?
process used to separate DNA and RNA fragments
describe the process of gel electrophoresis ?
. both DNA and RNA are negatively charged due to phosphate group
. they are run through a density gel
. shorter fragments migrate faster ( less resistance )
. use a ladder of known fragment sizes in one lane to be able to determine your DNA or RNA sizes
how can you visualize the DNA after gel electrophoresis ?
to visualize the DNA the gel is soaked in a dye ( ethidium bromide ) which binds in DNA and fluoresces under UV light
what is PCR ( polymerase chain reaction ) used for ?
. it can be used to determine genetic variation between DNA samples
. DNA sequence can be copied accurately and rapidly for analysis
. PCR can amplify target sequence 100 million fold in 2-3 hrs
what is the main ingredient used in PCR ?
. TaqDNA polymerase to copy DNA template using repeated cycles of replication
. it is derived from thermophilic bacterium
explain the steps of PCR reaction ?
- heat DNA ( 94-96) degrees to separate the DNA strands
- slight cooling of reaction mixture ( 50-65 ) degrees to allow the hybridization/annealing of primers ( oligonucleotide)
- extension heat to 72 deg and TaqDNA polymerase 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
what are the application of PCR ?
. provides numerous DNA templates for mutation screening
. forensic application
. mRNA templates can be analysed
. discriminate between DNA sequence differing by a single nucleotide (SNPs)
what is reverse transcription ?
RT-PCR can be used to check if mRNA is being transcribed and if protein can be translated
. reverse transcribe mRNA into cDNA using reverse transcriptase
what is chain length termination ?
. addition of ddNTP
.ddNTP lacks the 3’ -OH group and cannot form a phosphodiester bond which leads to chain termination
. requires incorporation of fluorescent labelled phosphate
.PCR reaction occurs
. 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
. we know the specific sequence of nucleotide within DNA
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
. one primer is the same for both reactions the other exits in two different versions
. primer A - is for normal sequence
. primer B - is for mutant sequence
explain the results for Allele-specific PCR ?
. TT = 220 bp
. CC = 205 bp
then its homozygous
. if one C and one T allele then one each 220 and 205 its homozygous
what is restriction fragment polymorphism ?
. restriction enzyme cut DNA
. 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
how to detect tandem repeats?
. use PCR or restriction enzyme to create DNA fragments to run on a gel electrophoresis and see how the variable number of tandem repeats- the more there is the bigger the fragment.
are mutations always bad ?
NO
they are the heart of evolution and natural selection
they can lead to better prognosis in disease
