molecular genetics

Question 1

why do we all have different phenotypic characteristics ?

Answer 1

due to variation which is genetic difference between individuals .

Question 2

what are the two types of variation ?

Answer 2

. polymorphisms

. disease causing mutations

Question 3

what are the two types of mutation ?

Answer 3

. point mutations

. frameshift mutations

Question 4

what is point mutation?

Answer 4

single nucleotide base is changed from a sequence of DNA or RNA due to a simple mistake during DNA replication in meiosis

Question 5

what are the consequence of point mutation?

Answer 5

often little consequence

e.g. sickle cell

Question 6

what is frameshift mutation?

Answer 6

caused by a deletion or insertion or inversion in a DNA sequence that shifts the way the sequence is read

Question 7

what is the consequence of frameshift mutation?

Answer 7

. more serious

Question 8

what percentage of population have polymorphisms ?

Answer 8

> 1%

unlikely to be cause of disease as it is abundent

Question 9

what is the consequence of polymorphisms?

Answer 9

can affect disease progression or drug response

Question 10

what are the 4 common types of polymorphism?

Answer 10

1. SNPs- single nucleotide polymorphism
2. Indels - small insertion/deletions
3. large scale copy number polymorphism - CNPs
4. tandem repeats

Question 11

what is the percentage of identical DNA between humans ?

Answer 11

99.9%

Question 12

what is the most common type of polymorphism?

Answer 12

SNPs

make up 80% of the 0.1% difference between individuals

Question 13

what type of mutation occurs in SNPs?

Answer 13

. point mutation

single nucleotide substitution of nucleotide with another

Question 14

where does SNPs occur?

Answer 14

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

Question 15

where is it more frequent for SNPs to occur ?

Answer 15

within non-coding region due to selection pressure

Question 16

what is property of SNPs?

Answer 16

most SNPs are neutral with no phenotype change

3-5% have a functional role and do not change phenotype

Question 17

what are the types of SNPs?

Answer 17

. 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

Question 18

what are indels?

Answer 18

. indels are insertion or deletions of nucleotide sequences of 2-10,000 base pairs
. second most common polymorphism

Question 19

what type of mutation occurs in indels?

Answer 19

frameshift mutation

Question 20

what is tandem repeats ?

Answer 20

. a sequence of nucleotide can be repeated numerous times in sequence

Question 21

what are the two types of tandem repeats ?

Answer 21

1. VNTRs - aka mini-satellites
   10-100 base long segments repeated
2. STRs - aka micro-satellite
   2-9 base long segments repeated

Question 22

what are CNPs?

Answer 22

entire gene repeated or deleted

Question 23

how is a polymorphism different from a mutation leading to a specific disease ?

Answer 23

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

Question 24

where do disease mutations often occur?

Answer 24

exons

Question 25

what are disease mutation referred to as ?

Answer 25

``` . point mutation . frameshift mutation . inversions . copy number variations mini and micro satellites are rare in exons ```

Question 26

what is non-disjunction?

Answer 26

. 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

Question 27

what are types of diseases that can occur due to non-disjunction?

Answer 27

. down's syndrome - 3 copies of chromosome 21 . edward's disease - 3 copies of chromosome 18 . patau's disease - 3 copies of chromosome 13

Question 28

effect of non-disjunction in sex chromosomes?

Answer 28

. turner's syndrome - females with only one x chromosome . kleinfelter's syndrome - XXY males . metafemale syndrome - XXX

Question 29

describe chromosome shape ? ( nomenclature )

Answer 29

Each human chromosome has a short arm "p" and long arm "q" separated by a centromere.

Question 30

what can we use to look at the structure and number of chromosomes ?

Answer 30

. karyogram which is then referred to as the karyotype

Question 31

what is karyotype and why is it used?

Answer 31

``` the process to look at chromosomal changes cab be used to compare . chromosome count . centromere position . banding pattern . sex chromosomes ```

Question 32

describe the process of karyotype?

Answer 32

``` . take cell sample . culture in dish . stop cell division during METAPHASE . extract chromosome from a single nucleus . stain with giemsa ```

Question 33

what are examples of molecular biology techniques used in lab to look at mutation ?

Answer 33

. DNA probe . restriction enzymes . gel electrophoresis

Question 34

what is DNA probe ?

Answer 34

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

Question 35

what is FISH ?

Answer 35

(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.

Question 36

what happens in FISH ?

Answer 36

. fluorescent DNA probes can be applied directly to chromosomes. . so the number of chromosomes per nuclei can be counted

Question 37

what are restriction enzymes ?

Answer 37

. 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

Question 38

what do restriction nucleases derive from ?

Answer 38

bacteria

Question 39

what is the difference between blunt ends and sticky ends ?

Answer 39

. 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

Question 40

what is gel electrophoresis ?

Answer 40

process used to separate DNA and RNA fragments

Question 41

describe the process of gel electrophoresis ?

Answer 41

. 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

Question 42

how can you visualize the DNA after gel electrophoresis ?

Answer 42

to visualize the DNA the gel is soaked in a dye ( ethidium bromide ) which binds in DNA and fluoresces under UV light

Question 43

what is PCR ( polymerase chain reaction ) used for ?

Answer 43

. 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

Question 44

what is the main ingredient used in PCR ?

Answer 44

. TaqDNA polymerase to copy DNA template using repeated cycles of replication . it is derived from thermophilic bacterium

Question 45

explain the steps of PCR reaction ?

Answer 45

1. heat DNA ( 94-96) degrees to separate the DNA strands 2. slight cooling of reaction mixture ( 50-65 ) degrees to allow the hybridization/annealing of primers ( oligonucleotide) 3. extension heat to 72 deg and TaqDNA polymerase forms a complimentary copy of the DNA strand

Question 46

what happens in the first 3 cycles of PCR ?

Answer 46

. 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

Question 47

what are the application of PCR ?

Answer 47

. provides numerous DNA templates for mutation screening . forensic application . mRNA templates can be analysed . discriminate between DNA sequence differing by a single nucleotide (SNPs)

Question 48

what is reverse transcription ?

Answer 48

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

Question 49

what is chain length termination ?

Answer 49

. 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

Question 50

what are some bio methods used to understand molecular genetics ?

Answer 50

. Allele-specific PCR - detecting point mutations . restriction fragment polymorphism analysis - detecting point mutation . detecting variable number tandem repeats

Question 51

what is allele specific PCR ?

Answer 51

. 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

Question 52

explain the results for Allele-specific PCR ?

Answer 52

. TT = 220 bp . CC = 205 bp then its homozygous . if one C and one T allele then one each 220 and 205 its homozygous

Question 53

what is restriction fragment polymorphism ?

Answer 53

. 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

Question 54

how to detect tandem repeats?

Answer 54

. 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.

Question 55

are mutations always bad ?

Answer 55

NO they are the heart of evolution and natural selection they can lead to better prognosis in disease