UNIT 2:  MOLECULAR GENETICS

 B.  Genetic Engineering  

 The principles of genetic engineering were experimentally established in the early 1970s.  Cohen and Boyer conducted a series of experiments that resulted in a method of selecting, recombining, and introducing new genes into bacteria via plasmid vectors.  By 1978, genetic engineering had commercial applications, producing the first human hormone, somatostatin.  These techniques are now used worldwide.  Genetic engineering serves many purposes today, such as in the production of widely used and very essential drugs that improve the quality of life (i.e. insulin and somatropin – see Figure 1, p. 294), and in the agricultural world, such as in improvements like the reduction of frost in crops by spraying them with a genetically engineered bacteria that prevent ice from crystallizing.

 Homework:  1-4, p. 295

 C.  Advanced Molecular Biological Techniques

 ·         since the discovery of r.e.s, DNA ligase, and plasmids, further advances have taken place in the field of genetic engineering, such as the polymerase chain reaction (PCR) technique

·         PCR is a direct method of making copies of a desired section of DNA

·         the methodology of PCR is related to the same process that takes place during DNA replication

·         instead using gyrase and DNA helicase to separate the double strands of the DNA from each other, PCR separates the strands from each other using heat (94ºC to 96ºC)

·         at these temperatures, the H-bonds break and the strands separate

·         in DNA replication, DNA polymerase III builds DNA from a 5’→3’ direction, and requires an RNA primer strand to begin the process

·         in PCR the RNA primers are replaced with DNA primers since they are easily synthesized in the laboratory

·         there are two DNA primers – each are complimentary to the target area to be copied

·         one of them is called a forward primer, the other is called a reverse primer because they mediate the synthesis of DNA in opposite directions toward each other

·         the temperature is brought down to the 50ºC to 65ºC range for the primers to anneal with the template DNA

·         once the primers have annealed, Taq polymerase, a DNA polymerase that is isolated from a bacterium that lives in hot springs, thus being an enzyme that can withstand high temperatures

·         Taq polymerase can build complementary strands using free nucleotides that have been added to a solution

·         the synthesis of the DNA strand takes place at a temperature of 72ºC – a temperature that is much too high for DNA polymerase III, which denatures above 37ºC

·         when the complimentary strand is made, the process repeats itself, doubling the number of strands each time

·         this results in the exponential increase of copies of the target DNA

·         after about 30 cycles, more than 1 billion copies of the targeted area will exist

·         the targeted area is not completely isolated in the first few cycles of DNA replication because Taq polymerase adds nucleotides until it reaches the end of the DNA, which is not necessarily the end of the target area

·         after the first cycle, variable-length strands are produced that start at the target region on the other end

·         in the second cycle, the DNA strands are again heated and separated, and the primers are allowed to anneal

·         on two of the DNA strands, one end terminates at the target region, and the primers anneal to the other end of the target area

·         Taq polymerase starts adding the appropriate nucleotides, commencing from the primer, and ceases when it reaches the end that terminates at the target region (see Figure 1, p. 297) – these strands are called constant-length strands

·         the remaining two strands are extended by Taq polymerase, as in the first cycle

·         by the third cycle, the number of copies of the targeted strands begins to increase exponentially

·         PCR is very practical since it only requires a small amount of DNA to work

·         it is useful in forensic criminal investigations, medical diagnoses, and genetic research

·         PCR has also been used by researchers to determine, from fossil remains, whether or not two species are closely related

·         for an animation of PCR click on http://medtech.cls.msu.edu/ISL/immunology/pcr.htm, or http://www.dnalc.org/shockwave/pcranwhole.html

 Homework:       1-5, p. 298

 D.  Restriction Fragment Length Polymorphism

 ·         any difference in DNA sequence that can be detected between individuals is called polymorphism

·         individuals of the same species carry genes coding for the same trait, but each possess a unique set of alleles for those genes

·         unless individuals are identical twins, then any two individuals of the same species are said to be polymorphic

·         polymorphism occurs in both coding and non-coding regions of DNA

·         in coding regions of DNA, polymorphisms can be useful when trying to identify individuals with mutation

·         in non-coding regions of DNA, polymorphisms can be useful when trying to identify an individual’s DNA makeup like in a forensic investigation – each individual possesses a specific number of tandem repeats, known as microsatellites

·         restriction fragment length polymorphism (RFLP) analysis involves the composition of different lengths of DNA fragments produced by a restriction enzyme digested and revealed by complementary radioactive probes to determine differences between any two individuals of the same species, or of different species

 THE PROCESS

• first the DNA is subjected to one or more r.e.s
• then the digested DNA is run on a gel, appearing as a long smear (see Figure 3, p. 299), because the amount of DNA is usually so large and the bands are so numerous that the fragments are unable to be resolved
• in order to distinguish DNA from two different sources, differences in the pattern that the fragments create must be detected, which is why the gel is then subjected to a chemical causing double-stranded DNA to be denatured into single-stranded DNA
• then a procedure known as Southern blotting is performed               

-          the single-stranded DNA is then transferred to a nylon membrane, with the aid of an electric current

-          the nylon membrane is placed on the gel with a positive electrode behind it

-          since DNA is negatively charged, it would transfer out of the gel and “blot” onto the nylon membrane, where it binds

-          the nylon membrane, containing the single-stranded DNA, is then immersed in a solution containing radioactive complementary nucleotide probes for either specifically chosen coding regions of the DNA, like the regions where mutations may occur, or specifically chosen non-coding regions of the DNA, like variable number tandem repeats (microsatellites)

-          wherever the complimentary sequences lie on the nylon membrane, complimentary base pairing will occur between the probes and the DNA – this is called hybridization

• when the nylon membrane is then placed against X-ray film, the radioactive probes cause exposure of the X-ray film in the areas where they have hybridized, called autoradiogram
• the film is then developed and the pattern is established
• this pattern can then be used for two basic purposes – to match a suspect’s DNA to the DNA found at a crime scene, or to detect a mutation that is believed to cause a genetic disorder
• to see how a DNA fingerprint is made, click on http://www.dnalc.org/resources/dnadetective.html
• the entire process is illustrated in Figure 3, p. 299, or you can click on http://www.dnalc.org/shockwave/southan.html or http://medtech.cls.msu.edu/ISL/immunology/rflp.htm

·         PCR and RFLP are both advanced molecular biological techniques that have benefited the field of genetic engineering considerably

·         a summary of each technique is outlined on p. 303, Table 2

·         some comparisons between the two can be made:

 

CRITERIA	RFLP Analysis	PCR
State of sample	·          large and fresh	·          minute (one cell big) and degraded
Size of sample	·          whole genome	·          target sequence only required
Time	·          three weeks	·          one day
Basic premise	·          cleaving DNA using restriction endonucleases followed by subjection to radioactive complementary DNA probes	·          building complementary strands using principles of DNA replication
Result medium	·          autoradiogram	·          gel
Tools	·          restriction endonucleases, radioactive probes, nylon membrane, X-ray film, gel electrophoresis	·          DNA polymerase, nucleotides (A, G, C, T), DNA primers, gel electrophoresis
Sensitivity and accuracy	·          highly sensitive and accurate	·          Sensitive and accurate

 

Homework:  6-14, p. 300

D.  DNA Sequencing

 

·         to completely analyze a particular gene’s structure and how it is related to its expression and specific polypeptide production, the exact sequence of base pairs for that gene must be determined

·         the Human Genome Project used computer technology to sequence and decipher the entire set of 46 chromosomes

·         the technology used was principally based on the gene sequencing lab techniques developed over the past 20 years

·         a method called the Sanger dideoxy method was developed in 1977 by Frederick Sanger and his colleagues

·         they determined the sequence of the an entire genome of a bacteriophage containing 5386 base pairs

·         the method developed by Sanger uses the principles of DNA replication – a process that requires the following:  a single stranded DNA template, a primer, DNA polymerase, and free nucleoside triphosphates

 

        THE PROCESS

 

·         the DNA template that is to be sequenced is treated so that it becomes single stranded

·         a short, single-stranded radioactively-labeled primer is added to the end of the DNA template

·         identical copies of the primed single-stranded DNA are placed in four reaction tubes, each containing DNA polymerase and a free nucleotides in the form of all four deoxynucleoside triphosphates, or dNTPs -- dATP, dTTP, dGTP, and dCTP

·         in each of these reaction tubes, complimentary strands are constructed (see Figure 4, p. 301)

·         each of the four reaction tubes also contains a different radioactively labeled dideoxy analogue in low concentrations –  this is a dNTP whose deoxyribose sugar is missing the – OH group on its 3’ carbon (see Figure 5, p. 302)

·         to see how dideoxy nucleotides are incorporated into DNA, click on http://bioweb.uwlax.edu/GenWeb/Molecular/Theory/DNA_sequencing/dnaseq.mov

·         for example, tube 1 may contain all four dNTPs plus small amounts of dideoxy-adenine (ddATP); tube 2, all four dNTPs plus dideoxy-thymine (ddTTP); tube 3, all four dNTPs pluc dideoxy-guanine (ddGTP), and tube 4, all four dNTPs plus dideoxy-cytosine (ddCTP)

·         DNA polymerase catalyzes the phosphodiester linkage between the 3’ – OH group of the deoxyribose sugar on the growing chain and the phosphate group on the incoming nucleotide – this means that if the 3’ – OH group is missing, DNA polymerase cannot add the next complementary base and synthesis stops

·         thus the dideoxy analogue that is incorporated into the chain acts as a chain terminator

·         this is why the Sanger method is also called the chain terminator technique

·         as a result, different lengths of complementary DNA will be built before the analogue is incorporated

·         for example, if the strand that is to be replicated consists of the sequence 3’ – AATGCATGCATTAGC – 5’, and it is part of the mix containing the dideoxy analogue adenine, four possible complimentary strands may be produced:

 

                                5’ – TTA                  5’ – TTACGTA                        5’ – TTACGTACGTA                              5’ – TTACGTACGTAA – 3’

 

·         as is seen above, the incorporation of the ddATP analogue blocks further growth of the chain

·         if a regular dATP, which is much higher in concentration than the ddATP analogue, incorporates itself into the complementary chain, elongation occurs

·         because the strands that are built will definitely differ in length, they can be separated by gel electrophoresis

·         at the end of replication, each tube is emptied into a different well on the gel

·         the sequence is then read off of the gel in ascending order (see Figure 6, p. 302)

·         after placing the gel against an X-ray film, the sequence of the nucleotides can be read from the autoradiogram

·         to see how this process is used to identify the mutation on a cystic fibrosis gene, click on http://www.library.csi.cuny.edu/~davis/molbiol/lecture_notes/protein_nucleicAnal/DNA_Sequencing_Figs.pdf

 

·         in the Human Genome Project the method was slightly different:

o        each ddNTP was fluorescently tagged – ddGTP was tagged green, ddATP yellow, ddTTP red, and ddCTP blue

o        there was only one reaction mixture (not four separate ones), each containing numerous copies of DNA to be sequenced, fluorescently tagged ddNTPs, and regular, untagged dNTPs

o        the fluorescently tagged ddNTPs incorporated themselves into the complementary, newly built strand

o        the sample was then electrophoresed through the gel

o        a computer read the sequence and the position of the tags on the gel, and a sequence of the DNA was determined

o        together with advances in computer technology, this technique deciphered the 3 billion-base-pair human genetic code

 

Homework:       16-22, p. 303