Molecular Biology Week 4

Question 1

Who devised the polymerase chain reaction?

Answer 1

Kary Mullis in the mid-1980s

Question 2

What did PCR enable?

Answer 2

It enabled the production of enormous numbers of copies of a specified DNA sequence.

Question 3

What is the principle of PCR?

Answer 3

This method is an in vitro enzymatic (catalyzed) method for amplifying exponentially a specific pre-selected fragment of DNA.

Question 4

What does PCR exploit in DNA replication? 2 points

Answer 4

DNA polymerase uses single stranded DNA as a template for the synthesis of a complementary new strand.
DNA polymerase requires a small section of double stranded DNA to initiate or “primer” synthesis.

Question 5

What are the four constituents involved in DNA amplification by PCR?

Answer 5

• Template is needed
  DNA polymerase enzyme (uses a single stranded DNA as template and uses a small section of the double stranded DNA to synthesise “primer” synthesis.
• Two synthetic
  oligonucleotide primers.
  -Four standard deoxyribosenucleoside triphosphates (dATP, dGTP, dTTP and dCTP)

Question 6

What are the three steps of PCR amplification?

Answer 6

(1) Denaturation; this is where a DNA template is first denatured by heat. This involbes strand separation of the double stranded DNA.
(2) Annealing; this is when specific oligonucleotides are annealed to two separate sites on opposite template strands.
(3) Extension; the extension of the primers by polymerase-mediated nucleotide additions to produce two copies of the original DNA sequence.

Question 7

How many times is this cyclic process repeated?

Answer 7

This process is repeated numerous times; to produce a double-stranded copy of the original DNA fragment defined by the oligonucleotide binding sites.

Question 8

What are the two types of DNA that are amplified?

Answer 8

One type is produced in the first PCR cycle :

1. This is where the original template is copied to generate a strand beginning at the 5’-end of the oligonucleotide primer and ending only when polymerase ceases to function.
2. A second type of product is defined at both the 5’ and 3’ ends as the syntehesis is terminated when the polymerase reaches the defined end of the template.

Question 9

After many cycles of PCR many copies of what are generated?

Answer 9

Many copies of a discrete double stranded fragment.

Question 10

How many temperature changes occur during the typical PCR protocol?

Answer 10

6, including the pre denaturation.

Question 11

What protein allows for the manipulation of DNA such as cloning?

Answer 11

Enzymes.

Question 12

Give examples of cloning vectors?

Answer 12

plasmids, bacteriophages (insertion and replacement), cosmids and yeast artificial chromosomes (for large pieces of DNA)

Question 13

Describe the template (source of DNA) used in PCR.

Answer 13

The amount of DNA needed is very small, less than a microgram of total DNA is sufficient. This DNA is often the total genomic DNA extracted from cells. PCR does not require purified DNA and is released by boiling the cells.

Question 14

How could DNA becom unstable?

Answer 14

By being exposed to nucleases

Question 15

What is an example of DNA that has been used/revised using PCR?

Answer 15

Human Papilloma Virus DNA, this had been detected in cervical carcinoma biopsies embedded in paraffin for over 40 years.

Question 16

What procedure is done to newborns for the detection of neonatal phenylketouria by PCR?

Answer 16

Blood samples are taken via heel prick and are stored as dried spots on cards for many years.

Question 17

Describe the original thermostable DNA polymerase used in PCR and one other heat stable DNA polyermase.

Answer 17

Originally e.coli, the enzyme was heat sensitibe and was being destroyed at the temps needed to separate double stranded DNA during PCR. Fresh had to be added each time. Now a heat stable polymerase was isolated from thermus aquaticus (Taq DNA polymerase). This bacterium lives in water at a temperature of 75C. Optima: 72C, however is stable even at 94C.

Question 18

Why is Taq polymerase used?

Answer 18

As it is heat stable and can be added only once at the start and will remain active, without being denatured throughout the process.

Question 19

What did Taq polymerase improve?

Answer 19

It improved the specificity and sensitivity of the PCR. Using Taq greatly reduced the non-specific binding of e.coli meaning that there is no amplification other than that of the target sequence.

Question 20

What is the disadvantage assocaited with using Taq polymerase?

Answer 20

In vitro Taq polymerase does not have a proofreading function and in a typical PCR, this enzyme can incorporate one incorrect nucleotide for about every 2 x 10^4 nucleotides added. Also produces a relatively small amount of amplicons. Proofreading thermostable polymerases can be bought however, though expensive.

Question 21

Describe the effect of Mg2+ ion concentration on the PCR?

Answer 21

Taq is sensitive to dialent cation concentrations. A conc of 2mM MgCl2 is thought to be optimal for most PCRs.
Anything higher is inhibitory to the polymerase.
Both the primers and the dNTPs (substrates for Taq) bind MG2+, the precise amount may need to be determined.

Question 22

Describe the effect that the buffer composition (KCl) can have in stimulating Taq DNA polymerase activity.

Answer 22

It can increase activity by up to 50-60% at 50mM. Anything higher than 75mM will inhibit activity.

Question 23

Name two PCR enhancers:

Answer 23

These enhancers improve PCR amplification of target sequences such as urea and formamide or dimethylsulfoxide.

Question 24

What are dNTPs?

Answer 24

These present in enxess as building blocks for DNA synthesis. A mix of all four is usually prepped. Are building blocks of DNA.

Question 25

How are concentrations of components of PCR mixtures usually calculated?

Answer 25

C1V1 = C2V2

Question 26

Why are primers used in PCR?

Answer 26

The sequence and combination of the primers determines the ultimate outcome of a PCR assay. Useful primer lengths are between 14 to 20 bases (up tto 40 can be used) in length with a 50% GC content.

Question 27

What are some of the general guidelines for primer design?

Answer 27

This is to max both efficiency and specificity in the amplification process:

• they should lie within conserved regions of the target genome.
• 3’ ends of the primers themselves should not be complementary, this avoids primer-diver generation.
• they must lack secondary structures
• both primers should have a matched G and C content, ensuring similar melting profiles.
• primers should be specific to a single member of a gene family.

Question 28

How is the annealing temperature for PCR calculated?

Answer 28

Tm = 4(G+C) + 2(A+T)C unit.

The tm for the primers should be within 5C of eachother.

Question 29

What are some probelms associated with PCR?

Answer 29

The slightest contamination of any equipment can result in the production of false positives.
Other problems result from the high error rate of the Taq polymerase, this can produce products that are not suitable for genetic analysis.
The technique also requires a certain amount of technical skill and some specified equipment to prep samples and perform effect PCRs.

Question 30

Name some variations of PCR?

Answer 30

Nested PCR, Hot start PCR, multiplex PCR, Reverse transcription.

Question 31

Describe nested PCR?

Answer 31

This can improve the sensitivity and specificity of a PCR.Two step method; amp of outer primer with target.
The second uses products of the first reaction. This second amp is then performed with an inner primer set. the product is very specific.

Question 32

What is Hot start PCR?

Answer 32

This technique reduced non-specific amplfication during the initial set up stages of the PCR. Can be performed by heating the reaction components to the tm before adding polymerase.

Question 33

Describe multiplex PCR:

Answer 33

This consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. Due to the multiple genes being targetted, additional info may be gained from a single test tun that would otherwhys require several times the reagents and time to perform.

Question 34

What is reverse transcriptiom PCR?

Answer 34

This is the amplifying of DNA from RNA. Reverse transcription of RNA into cDNA which is then amplified.Used in expression profiling to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites.

Question 35

Describe the role of PCR in diagnosis of diseases:

Answer 35

PCR permits the early diagnosis of malignant diseases such as leukemia and lymphomas. PCR assays can be performed directly on genomic DNA. PCR also permits the identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture assays and animal models. The amounf of viral load in a patient can also be quantified by PRC based DNA quantitaion techniques.

Question 36

What are genes composed of?

Answer 36

Genese are composed of sequences called exons and usually have introns.

Question 37

What are exons?

Answer 37

These contain the DNA sequence for the protein product of the gene.

Question 38

What are introns?

Answer 38

These can vary in number between 0-50. They are larger than the exon sequences and must be spliced out before the biological information can be used to synthesise a protein.

Question 39

What does alternative splicaing do?

Answer 39

This results in multiple proteins from a single gene.

Question 40

In the human genome, is categorized by?

Answer 40

It is categorized by fucntion of each gene product, given both as number of genes and percentage of all genes.