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Medical Cell Biology 🌟 > SESSION 12 > Flashcards

SESSION 12 Flashcards

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1
Q

Define restriction enzymes

A

Restriction enzymes also known as restriction endonuclease, are enzymes that cut a DNA molecule at a particular place (restriction site)
They are made by bacteria
They are also essential tool for recombinant DNA technology

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2
Q

Describe in general terms restriction analysis

Standard molecular process

A

Restriction endonuclease (restriction enzymes) are bacterial enzymes that recognise specific DNA sequences ‘restriction sites’ and cut the DNA

Cutting of the DNA sequence leaves ‘sticky ends’. This can be reversed, or different fragment joined, using DNA ligase

This together with electrophoresis, can be used to:

  • investigate the size of DNA fragments
  • investigate mutations (sickle cell disease)
  • investigate DNA variation (DNA fingerprinting)
  • gene cloning
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3
Q

Describe in general terms gene cloning

Standard molecular process

A

Plasmids are used from bacteria

  • small circular DNA
  • can transfer to other bacteria
  • can contain antibiotic genes

1) plasmid is cut using restriction enzymes, gene of interest is added to create ‘recombinant DNA’ molecule
2) this is introduced into bacterium- ‘transformation’
3) bacteria containing recombinant DNA are placed in environment to multiply

Plasmid often also contains an antibacterial resistance gene, in order to positively select for bacteria that have taken up the plasmid

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4
Q

Describe in general terms DNA sequencing- sanger dideoxy chain termination method
(Standard molecular process)

A

Fluorescent/ radioactively stained ddNTPs are added (with regular dNTPs) to DNA template strand along with DNA polymerase to create a complimentary DNA strand
DdNTPs are a variation of dNTPs lacking a 3’ OH group so polymerisation cannot occur. Therefore the strand terminates

Depending which ddNTP is used the new strand will stop at different places

This will produce lots of new DNA fragments of different lengths than can be denatured with heat and separated using gel electrophoresis. Each of the different ddNTP runs in a different land and the end result allows us to write the DNA sequence

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5
Q

Describe the theory behind DNA electrophoresis and how the technique can be sued t provide information about DNA fragments

A

DNA gel electrophoresis
Used to separate different sized DNA fragments:
1) a solution of different fragments is placed in a well at the negative anode end
2) a charge of the anodes encourages the (negatively charged) DNA to move towards the positive anode
3) larger fragments move slowest
4) fragments of known size are used as a reference

This requires: gel, buffer, power supply and stain

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6
Q

Why use restriction analysis?

A
  • to investigate the size of DNA fragments, e.g. Small deletions
  • to investigate mutations, e.g. Sickle cell disease
  • to investigate DNA variation, e.g. DNA fingerprinting
  • to clone DNA
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7
Q

Define plasmids

A
  • small circular dDNAs
  • found in bacteria
  • mini- chromosomes
  • carry genes to replicate independently
  • can transfer to other bacteria
  • often carry antibiotic resistance genes
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8
Q

Why clone human genes?

A
  • to make useful proteins, e.g. Insulin
  • to fins out what genes do, e.g. HTT
  • genetic screening, e.g. Huntington’s, BRCA1/2
  • genetic therapy, e.g. Cystic fibrosis
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9
Q

Explain PCR

A

Polymerase Chain Reaction (PCR)
PCR amplifies DNA segments by repeated copying of th target DNA using a thermo- stable DNA polymerase and a pair of primers that uniquely defien the region to be copied
The DNA polymerase is thermostable Taq DNA Polymerase from Thermus Aquaticus

1) denature at high temperatures (94- 96 degrees) single strands formed
2) renaturation (annealing) at lower temps (50-65 degrees)
3) DNA synthesis at medium temperature (75-80 degrees) extension- the bases complementary to the template are coupled to the primer on the 3’ side

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10
Q

Why use PCR?

A
  • to amplify a specific DNA fragment
  • to investigate single base mutations, e.g. Tay Sachs, sickle cell disease
  • to investigate small deletions or insertions, e.g. Cystic fibrosis
  • to investigate variation, genetic relationships, e.g. DNA profiling, DNA typing
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11
Q

Why is serum protein electrophoresis used?

A

Serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups similar size, shape and charge

Blood serum contains two major protein groups:
Alumni and globulin
They both carry substances through the bloodstream

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12
Q

Describe sodium dodecyl sulphate polyacrylamide gel electrophoresis and why it is used

A

Allows proteins to be separated purely on the basis of their molecular weight
The detergent SDS denatures protein molecules and gives the protein a negative charge proportionate to molecular weight
Electrophoresis then takes place with migration from negative to positive electrode, largest molecular weight travel further

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13
Q

Define isoelectric focusing

A

Proteins can be separated by their isoelectric point (pI)

Proteins are applied to a gel containing a pH gradient
A protein will migrate until it reaches a pH that matches its pI- at this point it will have no overall charge and so will stop migrating

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14
Q

What is two dimensional electrophoresis used for?

A

Allows the separation of complex mixtures of proteins that have identical pI values but different weights

The gel is turned by 90 degrees and ru for a different property to separate the bands out

Important for proteomics

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15
Q

Define proteomics

A

Proteomics is the large- scale study of proteins

  • digest protein with trypsin
  • perform mass spectrometry
  • generate list of peptide sizes
  • use database of predicted peptide size known protein to: identify protein
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16
Q

Define proteomics

A

Analysis of all proteins expressed form genome

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17
Q

Define molecular diagnosis

A

Analysis of a single purified protein

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18
Q

Define proteolysis

A

The breakdown of proteins or peptides into amino acids by the action of enzymes

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19
Q

Define DNA hybridisation

A

Refers to a molecular biological technique that measure the degree of genetic similarity between pools of DNA sequences

  • double stranded DNA denatures- hydrogen bonds break to form single strands
  • mixture cooled the single strands annel as hydrogen bonds can reform
  • DNA probe- single stranded DNA that is complementary to one strand, but labelled with radioactive markers
  • the single stranded DNA re-anneals to the probe
  • identified by photographic film
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20
Q

What are the different types of hybridisation techniques?

A

Southern blotting- uses DNA probes to identify complementary DNA sequences after gel electrophoresis

Northern blotting- uses DNA to detect RNA species

Western blotting- not a DNA hybridisation technique, involves the detection of proteins by antibodies after protein gel electrophoresis

21
Q

Describe Southern Blotting

A

The unlabelled DNA from gel electrophoresis can be marked during southern blotting
Nylon is used to transfer the fragments from electrophoresis
Gel is soaked in alkaline solution which denatures the DNA into single strands
Electrophoretic transfer- a voltage is applied across the set up
This is then hybridised with a labelled gene probe to show the specific DNA fragments
Use of radioactive probes can mark specific complimentary DNA fragment
Detect by exposure to X-ray film

22
Q

Why use southern blotting?

A
  • allows us to detect pieces of DNA from complex mixtures that would otherwise be very difficult
  • allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel

It is used in combination with PCR to detect:

  • gene structure, e.g. Large deletion
  • gene expansion, e.g. Triplet repeats
  • mutations in genetic tests, e.g. Sickle cell
  • genetic relationships, e.g. Fingerprinting
23
Q

Understand how PCR restriction analysis and DNA hybridisation can be used in allele- specific test

A

PCR- use primers specific for sequence either side of allele of interest to amplify specific allele

Restriction analysis- use one/ multiple restriction enzyme with restriction sites around/ within the allele. Analyse the size of fragments produced. If the restriction enzyme cuts the wild type but not sample, the restriction site is mutated/ missing

DNA hybridisation - use a DNA probe that is complementary to either the wild type or mutated allele

24
Q

What are the characteristics of probes?

A
  • probes are not 100% complementary to the sequence
  • probs do not have to complementary align with the target sequence
  • probes do not affect the position of the target sequence on a gel
25
Q

Define dideoxynucleotide triphosphate

A
  • very similar to the structure of deoxynucleotide triphosphate (DNA nucleotide bases)
  • it contains a H at 3’ position rather than an OH
  • it therefore blocks further elongation so no further phosphodiester bond will be able to be formed
26
Q

Define northern blotting

A

The northern blot is a technique to study gene expression by detection of RNA in a sample
It involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridisation probe complementary to part of the entire target sequence

The major difference between northern and southern blotting is that RNA, rather than DNA is analysed in the northern blot

27
Q

Describe the procedure of northern blotting

A
  • extraction of total RNA from a homogenised tissue sample or from cells
  • eukaryotic mRNA can then be isolated, to isolate only those RNAs with a poly(A) tail
  • RNA samples are then separated by gel electrophoresis, separating RNA by size
  • transferred to a nylon membrane through capillary action
  • probe has been labelled, it is hybridised to the RNA on the membrane
  • the membrane is washed to prevent background signals
  • hybrid signals are detected by X-ray film
28
Q

Describe RT- PCR (reverse transcriptase PCR)

A

A technique commonly used to detect RNA expression

RT- PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase
The newly synthesised cDNA is amplified using traditional PCR

  • taq polymerase does not recognise RNA
  • mRNA has a long poly (A) tail
  • therefore a primer is made with a long T chain with a 5’ prime end
  • a copy of cDNA is made -> enzyme reverse transcriptase
  • PCR amplifies the region within the primer
  • RT- PCR measures level of expression
29
Q

Define a microarray

A

A set of DNA sequences representing the entire set of genes of an rganism in a grid pattern on some sort of solid support for use in genetic testing

Hybridisation experiment -> label different patients DNA, and hybridise them onto the slide, basically measure, with fluorescent signal, how much of each DNA is binding to each part of the genome

Therefore you can assess the entire genome of an individual for changes in a copy number, for changes in expression

30
Q

Describe FISH(fluorescent in situ hybridisation)

A

Fluorescence in situ hybridisation uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementary

Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes

31
Q

Define chromosome painting

A

Refers to the hybridisation of fluorescently labelled chromosome- specific, composite probe pools to cytological preparations

32
Q

Where are restriction enzymes found?

A

Bacteria

33
Q

Define mutation

A

The changing of the structure of a gene, resulting in a variant from which may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion or rearrangement of larger sections of genes or chromosomes

34
Q

Explain the relationship between changes in nucleotide and amino acid sequences

A

A change in the genetic code can result in different amino acids being coded for:
E.g. AUA changed to AUG -> isoleucine changed to methionine.

A change in the primary sequence of a polypeptide can lead to a change in shape and therefore function of the protein

35
Q

Describe the different types of mutational changes, e.g. Point mutation, insertion and deletion

A

Point mutation
Base substitutions than can either:
- transition: purine -> purine or
Pyrimidine -> pyrimidine
- transversion: purine -> pyrimidine
Pyrimidine -> purine

Point mutations in the coding region of a protein can be a:

  • silent mutation
  • missense mutation
  • nonsense mutation

Point mutations in non- coding regions or outside genes can of course also be detrimental as they can alter binding sites, promotor sequence, splice sites

Insertions and deletions
A sequence is added or removed from the nuclei acid. It can be a single nucleotide, a few nucleotides or millions of nucleotides
Insertions/ deletions can cause frameshift mutation

36
Q

Predict and explain the effect that different frameshift mutations may have

A

Silent mutation:
A mutation that does not alter the amino acid specified
E.g. CUA(leu) -> CUG (leu)

Missense mutation
A mutation that replaces one amino acid with another
E.g. GAC (Asp) -> GAA (Glu)

Nonsense mutation
A mutation that change the amino acid specified to a stop codon
E.g. UGG(Trp) -> UGA(stop)

Frameshift mutation
Addition or subtraction of nucleotides not in multiples of 3
E.g. A single base insertion of C that shifts all following triplet codes up one nucleotide

37
Q

Describe how spontaneous and induced mutations may occur

A

Spontaneous mutations

  • not caused by exposure to a known mutagen
  • errors in DNA replication
  • DNA bases have slight helical instability

The rate of spontaneous mutations differed for different genes:
- dependent on size
- dependent on sequence
Each individual has multiple new mutations and must by chance are not in the coding regions

Induced mutations

  • chemicals and radiation can cause mutations
  • chemicals that cause a mutation are called mutagens, they’re mutagenic
  • chemicals that cause cancer are called carcinogens, they’re carcinogenic

Examples:

  • alkylation agents- remove a base
  • acridine agents- add or remove a base
  • X-rays- break chromosomes/ delete few nucleotides
  • UV radiation- creates thymidine dimers
38
Q

Define mutation

A

Mutation- ‘a change in a nuclear acid sequence, which can be the addition of one or more nucleotides, the removal of one or more nucleotides, or the rearrangement of several nucleotides

39
Q

Define wild type

A

An individual within a population displaying a wild- type trait, which is the trait that is most common in that population

  • a mutation causes a mutant phenotype, which is a phenotype that differs from the common or wild type phenotype in the population
  • a mutation in a gene causes a mutant allow, which is an allele that differs from the common allele in the population
  • mutations that occur in the germ line have the possibility of being passed n to offspring- germ line mutations
40
Q

Describe the process and role of DNA repair

A

Mutations happen very frequently but are being recognised and repaired very frequently too

Mismatch repair
This occurs when enzymes detect nucleotides that odn’t base pairs in newly replicated DNA
The incorrect base paired is excised and replaced. Detection of mismatches is termed proofreading

Excision repair
Damaged DNA is removed by excision of base paris and replacement by DNA polymerase

Nucleotide excision
Repair replaces up to 30 bases and is used in repair of UV damage and some carcinogens

Base excision repair
Replaces 1-5 bases and repairs oxidative damage

When DNA repair fails an increased number of mutations will be present in the genome
The protein p53 monitors repair of damaged DNA and if damage is too severe it promotes apoptosis

41
Q

Explain the relationship between DNA damage and cancer

A

If DNA is damaged to the extent that apoptosis doesn’t occur or the damage leads to uncontrolled growth the cancerous cells are produced

Tumours:

  • tumours are derived from individual abnormal cells
  • they arise from th lack of normal growth control
  • generated by a multistep process
  • tumours are more likely to arise from cell types undergoing frequent cell divion
  • all of the cells in a tumour are of the same type
  • the behaviour of a tumour depends on the cell type
Oncogenes:
Genes involved in control of cell division 
- they are present in normal cels
- many different classes
- may stimulate/ inhibit growth

Tumour suppressor genes
genes involved in protecting the cell against one step on the path to cancer

When the genes are normally present in cells they’re Porto- oncogenes
It is after that mutation that a proto- oncogene becomes an oncogene
Viruses can carry copies of oncogenes, the presence of a virus means that the gene doesn’t function as normal, e.g. HIV

42
Q

Recognise the fundamental importance of PCR in the diagnosis of genetic disease

A

Most human mutations are single base changes and therefore hard to detect

PCR amplifies DNA segments by repeated copying, using a thermo- stable DNA polymerase and a pair of primers that uniquely define the region to be copied
1) DNA is denatured- @95degrees for 1 min
2) Anneal the primers- @55degrees for 1 min
3) copy the DNA- @72degrees for 2 min
(Taq DNA Polymerase)
Not all mutations result in the loss/ gain of a restriction site:
/\F508 mutation Cystic Fibrosis
3 base deletion (no frameshift)
Loss of phenylalanine

PCR amplification and southern blotting

43
Q

Provide an overview of the different genetic tests available for the detection of mutations in genes

A

Southern blotting
This is the technique of choice when there is a need to analyse larger segments of DNA within and around a gene
Southern blotting is also used to analyse triplet repeat disorders such as Huntington’s disease and Fragile X syndrome

Array Comparative Genomic Hybridisation (Array CGH)
This is used to screen for sub- microscopic chromosomal deletions for which the location cannot be deduced from the patient’s phenotype
1) an array of DNA probes covering the entire genome is applied to the surface of a solid matrix
2) patient DNA and normal control DNA are each labelled with different coloured flurescent tags, e.g. Patients DNA labelled red and normal DNA labelled green
3) equal amounts of the labelled DNA are then hybridised to the probe area and the hybridisation signals are detected and compared
4) for probes where the signal of the normal DNA exceeds that of the patient’s DNA, the patient has a deletion of the chromosomal region from which that probe was derived

44
Q

Show an appreciation of the ethical issues associated with genetic testing

A

Example:
If patient X get’s tested for a dominant X-linked genetic disease because their grandmother suffered from the disease and tests positive. Patients X’s mother must have the genetic disorder but may not have expressed the symptoms yet.
Is it right to tell the mother, as she is likely to show signs and symptoms of the disease sooner than patient X?

Genetic testing is used prenatal also and used in abortion cases

45
Q

Describe numerical and structural chromosome abnormalities and their significance

A

Numerical- a number of chromosomes other than 46
Polyploidy (e.g. Triploidy or tetraploidy)
Aneuploidy (an abnormal number that is not a multiple of the haploid number)
- Monosomy is a loss of one hero some, i.e. One chromosome pair exists as a single chromosome
- trisomy is a gain of one chromosome, i.e. One chromosomes pair exists as a triplet

Structural- physical changes to one or more of the chromosomes

  • balanced, when the change does not cause any missing or extra genetic information
  • unbalanced, when the changes cause missing or extra genetic information
46
Q

Describe chromosome mutations within one chromosome

A

Deletion- loss of genetic infromation

Duplication- some genetic material is doubled

Inversion- no loss of genetic material, but a rearrangement of genetic material

Ring chromosomes- loss of telomeres or ends of both arms and formation of a ring

Isochromosome- creation of two non- identical chromosomes, one is a combination of the two short arms, the other is a combination of the two long arms

47
Q

Describe chromosome mutations with two chromosomes

A

Inversion- no lord of geneti mateial, but as rearrangement of genetic material to a non- homologous chromosome

Reciprocal translocation- no loss of genetic material, but an exchange of genetic material between tow non- homologous chromosomes

Robertsonian translocation- rearrangement of genetic material between two chromosome; the q arms (long) of two acrocentric chromosomes combine to form one ‘super chromosome with the loss of both p- arms

48
Q

Be familiar with the concept of karyotyping

A

Karyotypes can be produced by cut&paste of chromosomes pictures into a systemically organised set of metaphase chromosomes organised in paris

Chromosome 1 is the largest chromosomes and chromosome 22 is the smallest
Chromosomes are numbered and grouped according to their size and the position of their centromere
The 23rd pair is the sex chromosomes

Medical Cell Biology 🌟 (15 decks)

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