DNA Technology and Genomics - Comprehensive Notes

Noncoding DNA

  • On average, about 99.9 percent of the DNA between two humans is the same, with the remaining percentage making us unique.
  • Humans, like most complex eukaryotes, have a huge amount of noncoding DNA, about 97% of the total.
  • Noncoding DNA consists of:
    • Gene control sequences such as promoters and enhancers.
    • Introns (whose total length may be ten times greater than the exons of a gene).
    • DNA located between genes consisting of repetitive DNA nucleotide sequences. Such sequences are found at the centromeres and ends of chromosomes.
    • The repetitive DNA at chromosome ends are called telomeres and have a protective function; a significant loss of telomeric DNA quickly leads to cell death. Abnormal lengthening of this DNA may help “immortal” cancer cells evade normal cell aging.

Steps in DNA identification

  • Copying DNA: Polymerase Chain Reactions (PCR)
  • Cutting DNA: Restriction Enzymes
  • Sorting DNA by size: Gel Electrophoresis
  • Comparing DNA: DNA fingerprinting

The PCR method

  • The PCR method is used to amplify DNA sequences.
  • When the source of DNA is scanty, impure, or in a partially degraded state, the polymerase chain reaction, or PCR method is used for preparing large quantities of a particular gene. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified.
  • Using this technique, any specific target segment within a DNA molecule can be quickly amplified (copied many times) in a test tube. By amplifying that DNA, it allows us to study that DNA molecule in detail in the laboratory.
  • Starting with a single DNA molecule, automated PCR machines called thermocyclers can generate 100 billion similar molecules in a few hours - enough DNA for restriction fragment analysis or other DNA technologies.
  • However since errors may occur during PCR replication, PCR cannot replace gene cloning in cells when large amounts of DNA are needed.

The PCR method - Steps

  1. A DNA sample is mixed with a heat tolerant DNA replication enzyme - DNA polymerase, DNA nucleotides, and primers that are complementary to the ends of the DNA fragment that is to be copied. (Primers are artificially made pieces of single-stranded DNA that are 20 - 30 nucleotides long that must be present for DNA polymerase to initiate replication).
  2. The solution is then exposed to cycles of heating (to separate the DNA strands) and cooling.
  3. During each cycle, the DNA is replicated, doubling the amount of DNA.
  4. The key to automating PCR was the discovery of an unusual heat-stable DNA polymerase, first isolated from prokaryotes living in hot springs in Yellowstone National Park. Unlike most proteins, this enzyme can withstand the heat at the start of each cycle.

PCR - Detailed explanation

  • In the polymerase chain reaction (PCR), a scientist chooses a DNA fragment to copy and designs primers that will bind to both ends of the fragment. DNA polymerase copies the segment between the two primers.
  • Repeating the procedure through about 30 cycles generates millions of copies of a single piece of DNA fragment.

DNA amplification by PCR

  • During each PCR cycle, the target DNA sequence is doubled. By the end of the third cycle, one-fourth of the molecules correspond exactly to the target sequence, with both strands of the correct length.
  • After 20 or so cycles, the target sequence molecules outnumber all others by a billionfold or more.

Cutting DNA - Restriction enzymes

  • Restriction enzymes cut DNA at specific sequences
  • To cut long DNA molecules into short pieces, biologists use bacterial proteins called restriction enzymes. (Restriction enzymes are naturally found in bacteria as a defense mechanism to cut up foreign DNA.)
  • There are 100s of different restriction enzymes – each enzyme is very specific. Each restriction enzyme only recognizes a particular short DNA sequence (usually four to eight nucleotides long). The restriction enzyme cuts both DNA strands of the double helix at specific points within the sequence producing pieces of DNA called restriction fragments.
  • The cuts may be staggered, yielding 2 double-stranded DNA fragments with single- stranded overhanging ends, called “sticky ends.”
  • For example, the two "sticky" ends originating from 2 different sources (GATGC and CTACG) compliment each other:
    • 5'-ATCTGACTGATGCGTATGCT-3'
    • 3'-TAGACTGACTACGCATACGA-5'
  • Sticky ends are the key to joining DNA restriction fragments originating from different sources.
  • These short extensions can form hydrogen-bonded base pairs with complementary single-stranded stretches of DNA
  • To add a piece of DNA from another source, the DNA from the other source is also cut using the same restriction enzyme. Hence the DNA from the other source also has identical sticky ends

Gel electrophoresis sorts DNA molecules by size

  • Gel electrophoresis uses a gel (a thin slab of jellylike material) as a molecular sieve to separate nucleic acids or proteins on the basis of size or electrical charge.
  • To separate DNA molecules in different mixtures:
    1. Restriction enzymes are used to prepare DNA fragments in each mixture.
    2. A sample of each mixture is placed in a well at one end of a flat, rectangular agar gel slab.
    3. A negatively charged electrode from a power supply is attached near the DNA-containing end of the gel, and a positive electrode is attached near the other end.
    4. Because DNA molecules have negative charge owing to their phosphate groups, they all travel through the gel toward the positive pole.
    5. As they move, the polymer fibers within the gel slows down the movement of the longer molecules more than it does shorter ones, separating them by length.
    6. After about ½ hour the electrodes are disconnected.
    7. Thus, gel electrophoresis separates a mixture of DNA molecules into bands, each band consisting of DNA molecules of the same length, with shorter molecules toward the bottom.
    8. The differences in restriction fragments produced in this way are called restriction fragment length polymorphisms (RFLPs, pronounced “rif-lips”).

Use of electrophoresis for DNA fingerprinting

  • Identical twins have identical DNA
  • Although only 1% of our DNA makes us unique, it means that there are around three million base pairs that are different between two people. These differences can be compared and used to help distinguish you from someone else.
  • Electrophoresis allows us to see:
    1. Similarities as well as differences between mixtures of restriction fragments belonging to the same individual
    2. Similarities as well as differences between the base sequences in DNA from two individuals.

Using restriction enzymes & gel electrophoresis to differentiate DNA

  • DNA to be analyzed is collected
  • Restriction enzymes are used to cut the DNA molecule into different sizes
  • Gel Electrophoresis sorts the DNA molecule fragments by size
  • Example: Let’s imagine that the a DNA sample was collected at a crime scene & another sample was collected from a suspect.
  • The 2 DNA sequences differ by a single base pair (highlighted in gold).
  • The restriction enzyme used cut the DNA between two cytosine (C) bases in the sequence CCGG & in its complement, GGCC.
  • Because the crime scene DNA has 2 recognition sequences for the restriction enzyme, it is cleaved in 2 places, yielding 3 restriction fragments (labeled w, x, & y).
  • DNA from the suspect, however, has only 1 recognition sequence & yields only 2 restriction fragments (z, & y).
  • In the 2 samples, the lengths of restriction fragments, & their numbers, differ depending on the exact sequence of bases in the DNA

Making a DNA Fingerprint

  • To permanently preserve the DNA fragments that are isolated by gel electrophoresis, the pieces of DNA are transferred or ‘blotted’ out of the fragile gel onto a nylon membrane.
    1. A positively charged nylon membrane is placed over the gel and the negatively charged DNA fragments are transferred to membrane
    2. DNA is then ‘unzipped’ to produce single strands of DNA.
    3. To visualize specific DNA pieces, biologists incubate the nylon membrane with radioactive probes. They can prepare a nucleic acid probe complementary to the DNA of interest and label it radioactively.
    4. A sheet of X-ray film placed over the gel will be exposed only where the desired DNA is on the gel. The resulting pattern of bands is called a DNA fingerprint.
    5. To compare two or more different DNA fingerprints the different DNA samples are run side-by-side on the same electrophoresis gel.

Preparing Nucleic Acid Probes

  • Probes are small fragments of minisatellite DNA tagged with radioactive phosphorous.
  • When at least part of the nucleotide sequence of a gene is already known or can be guessed, this information can be used to synthesize a short single strand of DNA with the complementary sequence and label it with a radioactive isotope or fluorescent dye.
  • This labeled, complementary single-stranded nucleic acid molecule is called a nucleic acid probe and is used to find a specific gene or other nucleotide sequence within a mass of DNA. The probe hydrogen-bonds to the complementary sequence in the targeted DNA.
  • This method is used for detecting genes/specific DNA pieces depend on base pairing between the gene/DNA piece and a complementary single strand sequence on another nucleic acid molecule, either DNA or RNA.

How a DNA probe tags a gene by base pairing

  • Use of a nucleic acid probe: Once the researcher identifies a colony carrying the desired gene, the cells can be grown further and the gene of interest (and/or its protein product) isolated in large amounts.

Nucleic Acid Probe Hybridization

  • Hybridization with a complementary nucleic acid probe detects a specific DNA within a mixture of DNA molecules. In this example, a collection of bacterial clones (colonies) are screened to identify those carrying a plasmid with a gene of interest.

Accuracy of DNA Fingerprints

  • A DNA fingerprint is a powerful identification tool. Minisatellites are short sequences (10-60 base pairs long) of repetitive DNA that show greater variation from one person to the next than other parts of the genome. This variation is exhibited in the number of repeated units or ‘stutters’ or VNTRs in the minisatellite sequence.
  • Analyzing only one VNTR is like looking at only one digit of a telephone number.
  • DNA fingerprinting typically compares from 5 to 13 VNTR loci.
  • DNA fingerprinting is a technique that simultaneously detects lots of minisatellites in the genome to produce a pattern unique to an individual.
  • 13 VNTR loci often used by some crime scene labs in their crime scene profiles, produce the odds that 2 persons will share a DNA profile at about 1 in 100 billion. There are about 6.5 billion people on the earth, so this should be enough to remove any doubt.

Variable Number Tandem Repeats (VNTR)

  • The non-coding DNA segments may vary in their length and they are called length polymorphisms.
  • Some length polymorphisms are made up of short repeating sequences of DNA nucleotides. These nucleotides can repeat few or many times in tandem (one after another) and are called variable number tandem repeats (VNTR).
  • The number of tandem repeats at a specific loci in DNA vary among individuals and are used for DNA identification.

Steps of DNA Fingerprinting

  1. DNA sample Extraction
  2. Restriction enzymes
  3. Electrophoresis
  4. Transfer to membrane
  5. Incubation with labelled probes
  6. X-ray