In Figure 10.6, a dotted box is found around a stretch of cytosine nucleotides in both the HV1 and HV2 regions. These regions are commonly referred to as 'C-stretches'. On the Anderson reference sequence that is shown in Figure 10.6, the HV1 C-stretch spans nucleotides 16184—16193 with a T at position 16189. In some samples position 16189 is a C giving rise to a stretch of 10 or more cytosines in a row (see Figure 10.7). The HV2 C-stretch region spans positions 303-315 on the reference sequence with a T at position 310 (Figure 10.6). This T can become a C in some samples leading to a homo-polymeric C-stretch.
Unfortunately, this homopolymeric stretch of cytosines creates problems for polymerases as they synthesize a complementary strand to the mtDNA template present in the reaction. Length heteroplasmy in HV1 between positions 16184 and 16193 can result in C-stretch lengths ranging from 8-14 cytosine residues (Bendall and Sykes 1995). Length heteroplasmy likely results from replication slippage after a T to C transition has occurred at position 16189. The mixture of length variants may already be present in the original DNA or generated in the sequencing reaction itself. Regardless of the source of the length variants, the impact of a 16189 T to C transition on sequencing results downstream of the C-stretch region can be seen in Figure 10.7.
A similar situation occurs with the HV2 C-stretch region when insertions of cytosines occur in the 303-310 area or a transition of T to C occurs at position 310 (Stewart et al. 2001). The presence of intra-individual variation in the number of cytosines observed when multiple hairs were tested from the same individual has led to the decision to not call an exclusion based solely on differences in the HV2 C-stretch region (Stewart et al. 2001). The issue of heteroplasmy and intra-individual variation will be discussed in more detail later in this chapter.
The ability to rapidly screen for the C-stretch prior to sequencing is advantageous and can be performed by noting the presence of extra heteroduplex peaks in quality control analyses of HV1 PCR products (Butler et al. 1998a).
Figure 10.6 (on following two consequent pages) Annotation of the revised Cambridge Reference Sequence for mtDNA control region with primer positions and common sequence polymorphisms examined in screening assays (see Figure 10.10).
AATACCAACT ATCTCCCTAA TTGAAAACAA TTATGGTTGA TAGAGGGATT AACTTTTGTT 15850 15860 15870
AATACTCAAA TGGGCCTGTC CTTGTAGTAT TTATGAGTTT ACCCGGACAG GAACATCATA 15880 15890 15900
AAACTAATAC TTTGATTATG 15910
GAAAAAGTCT CTTTTTCAGA 15970
ACCAGTCTTG TAAACCGGAG ATGAAAACCT TGGTCAGAAC ATTTGGCCTC TACTTTTGGA 15920 15930 15940
TTAACTCCÄC CATTAGCACC CAAAGCTAAG AATTGAGGTG GTAATCGTGG GTTTCGATTC
TTTTCCAAGG ACAAATCAGA AAAAGGTTCC TGTTTAGTCT 15950 15960
ATTCTAATTT AAACTATTCT TfiAGATTAAA TTTGATAAGA
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This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.